US20160130315A1 - Tumor tissue-penetrating peptide specific to neuropilin and fusion protein having same peptide fused therein - Google Patents
Tumor tissue-penetrating peptide specific to neuropilin and fusion protein having same peptide fused therein Download PDFInfo
- Publication number
- US20160130315A1 US20160130315A1 US14/893,317 US201414893317A US2016130315A1 US 20160130315 A1 US20160130315 A1 US 20160130315A1 US 201414893317 A US201414893317 A US 201414893317A US 2016130315 A1 US2016130315 A1 US 2016130315A1
- Authority
- US
- United States
- Prior art keywords
- neuropilin
- peptide
- tumor tissue
- tpp
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 217
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 179
- 108050009450 Neuropilin Proteins 0.000 title claims abstract description 114
- 102000002111 Neuropilin Human genes 0.000 title claims abstract description 113
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 26
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 26
- 230000027455 binding Effects 0.000 claims abstract description 157
- 201000011510 cancer Diseases 0.000 claims abstract description 50
- 230000033115 angiogenesis Effects 0.000 claims abstract description 37
- 239000002502 liposome Substances 0.000 claims abstract description 28
- 201000010099 disease Diseases 0.000 claims abstract description 25
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 25
- 230000036952 cancer formation Effects 0.000 claims abstract description 21
- 239000002105 nanoparticle Substances 0.000 claims abstract description 21
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 18
- 229940126586 small molecule drug Drugs 0.000 claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 53
- 235000018102 proteins Nutrition 0.000 claims description 50
- 102000004169 proteins and genes Human genes 0.000 claims description 50
- 150000001413 amino acids Chemical group 0.000 claims description 45
- 102000014105 Semaphorin Human genes 0.000 claims description 31
- 108050003978 Semaphorin Proteins 0.000 claims description 31
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 30
- 239000000427 antigen Substances 0.000 claims description 27
- 108091007433 antigens Proteins 0.000 claims description 27
- 102000036639 antigens Human genes 0.000 claims description 27
- 239000012634 fragment Substances 0.000 claims description 27
- 230000004927 fusion Effects 0.000 claims description 27
- 239000000203 mixture Substances 0.000 claims description 20
- 235000001014 amino acid Nutrition 0.000 claims description 18
- 108091033319 polynucleotide Proteins 0.000 claims description 13
- 102000040430 polynucleotide Human genes 0.000 claims description 13
- 239000002157 polynucleotide Substances 0.000 claims description 13
- 102000005962 receptors Human genes 0.000 claims description 12
- 108020003175 receptors Proteins 0.000 claims description 12
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 6
- 230000001404 mediated effect Effects 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 4
- 102000014914 Carrier Proteins Human genes 0.000 claims description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 3
- 235000004279 alanine Nutrition 0.000 claims description 3
- 108010078791 Carrier Proteins Proteins 0.000 claims description 2
- 102000004127 Cytokines Human genes 0.000 claims description 2
- 108090000695 Cytokines Proteins 0.000 claims description 2
- 102000003886 Glycoproteins Human genes 0.000 claims description 2
- 108090000288 Glycoproteins Proteins 0.000 claims description 2
- 102000018697 Membrane Proteins Human genes 0.000 claims description 2
- 108010052285 Membrane Proteins Proteins 0.000 claims description 2
- 108010085220 Multiprotein Complexes Proteins 0.000 claims description 2
- 102000007474 Multiprotein Complexes Human genes 0.000 claims description 2
- 108091058545 Secretory proteins Proteins 0.000 claims description 2
- 102000040739 Secretory proteins Human genes 0.000 claims description 2
- 102000040945 Transcription factor Human genes 0.000 claims description 2
- 108091023040 Transcription factor Proteins 0.000 claims description 2
- 108010067390 Viral Proteins Proteins 0.000 claims description 2
- 239000005556 hormone Substances 0.000 claims description 2
- 229940088597 hormone Drugs 0.000 claims description 2
- 102000035118 modified proteins Human genes 0.000 claims description 2
- 108091005573 modified proteins Proteins 0.000 claims description 2
- 230000007659 motor function Effects 0.000 claims description 2
- 230000031787 nutrient reservoir activity Effects 0.000 claims description 2
- 108091006024 signal transducing proteins Proteins 0.000 claims description 2
- 102000034285 signal transducing proteins Human genes 0.000 claims description 2
- 239000003053 toxin Substances 0.000 claims description 2
- 231100000765 toxin Toxicity 0.000 claims description 2
- 108700012359 toxins Proteins 0.000 claims description 2
- 102000035160 transmembrane proteins Human genes 0.000 claims description 2
- 108091005703 transmembrane proteins Proteins 0.000 claims description 2
- 108060003951 Immunoglobulin Proteins 0.000 claims 1
- 239000003102 growth factor Substances 0.000 claims 1
- 102000018358 immunoglobulin Human genes 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 35
- 238000000034 method Methods 0.000 abstract description 35
- 230000008595 infiltration Effects 0.000 abstract description 28
- 238000001764 infiltration Methods 0.000 abstract description 28
- 230000001965 increasing effect Effects 0.000 abstract description 23
- 210000003556 vascular endothelial cell Anatomy 0.000 abstract description 19
- 206010015866 Extravasation Diseases 0.000 abstract description 10
- 230000036251 extravasation Effects 0.000 abstract description 10
- 238000001727 in vivo Methods 0.000 abstract description 10
- 230000001093 anti-cancer Effects 0.000 abstract description 3
- 230000008685 targeting Effects 0.000 abstract description 3
- 230000002491 angiogenic effect Effects 0.000 abstract 1
- 230000000437 effect on angiogenesis Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 142
- 210000001519 tissue Anatomy 0.000 description 83
- 102000004207 Neuropilin-1 Human genes 0.000 description 79
- 108090000772 Neuropilin-1 Proteins 0.000 description 79
- 102000004213 Neuropilin-2 Human genes 0.000 description 72
- 108090000770 Neuropilin-2 Proteins 0.000 description 72
- 239000013598 vector Substances 0.000 description 55
- 102000009524 Vascular Endothelial Growth Factor A Human genes 0.000 description 53
- 108010090319 Semaphorin-3A Proteins 0.000 description 52
- 102000013008 Semaphorin-3A Human genes 0.000 description 47
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 42
- 238000002474 experimental method Methods 0.000 description 42
- 239000003446 ligand Substances 0.000 description 35
- 229960005395 cetuximab Drugs 0.000 description 34
- 229960000575 trastuzumab Drugs 0.000 description 32
- 241000282414 Homo sapiens Species 0.000 description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 28
- 210000004204 blood vessel Anatomy 0.000 description 24
- 230000035699 permeability Effects 0.000 description 23
- 102000000905 Cadherin Human genes 0.000 description 21
- 108050007957 Cadherin Proteins 0.000 description 21
- 230000010261 cell growth Effects 0.000 description 20
- 239000000126 substance Substances 0.000 description 20
- 230000014509 gene expression Effects 0.000 description 18
- 241000699670 Mus sp. Species 0.000 description 17
- 229940024606 amino acid Drugs 0.000 description 17
- 238000004458 analytical method Methods 0.000 description 17
- 238000010586 diagram Methods 0.000 description 16
- 238000003364 immunohistochemistry Methods 0.000 description 16
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 14
- 239000003814 drug Substances 0.000 description 14
- 239000011780 sodium chloride Substances 0.000 description 14
- 238000001262 western blot Methods 0.000 description 14
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 13
- 102000008790 VE-cadherin Human genes 0.000 description 13
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 13
- 108010018828 cadherin 5 Proteins 0.000 description 13
- 230000001976 improved effect Effects 0.000 description 13
- 230000006872 improvement Effects 0.000 description 13
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 13
- 229910019142 PO4 Inorganic materials 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 235000021317 phosphate Nutrition 0.000 description 12
- 239000010452 phosphate Substances 0.000 description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 12
- 108050009312 plexin Proteins 0.000 description 12
- 102000002022 plexin Human genes 0.000 description 12
- 238000000746 purification Methods 0.000 description 12
- 241001123946 Gaga Species 0.000 description 11
- 108010034265 Vascular Endothelial Growth Factor Receptors Proteins 0.000 description 11
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 11
- 238000003556 assay Methods 0.000 description 11
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 11
- 230000008728 vascular permeability Effects 0.000 description 11
- 239000000872 buffer Substances 0.000 description 10
- 230000008859 change Effects 0.000 description 10
- 238000012790 confirmation Methods 0.000 description 10
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 10
- 238000010494 dissociation reaction Methods 0.000 description 10
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 10
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 10
- 229940124597 therapeutic agent Drugs 0.000 description 10
- 238000002965 ELISA Methods 0.000 description 9
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 9
- 102000058223 human VEGFA Human genes 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 9
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 9
- 239000002773 nucleotide Substances 0.000 description 9
- 125000003729 nucleotide group Chemical group 0.000 description 9
- 230000019491 signal transduction Effects 0.000 description 9
- 210000004881 tumor cell Anatomy 0.000 description 9
- 229920001213 Polysorbate 20 Polymers 0.000 description 8
- 108020005091 Replication Origin Proteins 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 201000010536 head and neck cancer Diseases 0.000 description 8
- 208000014829 head and neck neoplasm Diseases 0.000 description 8
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 8
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 8
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 7
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 7
- 101150029707 ERBB2 gene Proteins 0.000 description 7
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 7
- 102000004961 Furin Human genes 0.000 description 7
- 108090001126 Furin Proteins 0.000 description 7
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 7
- 241000699660 Mus musculus Species 0.000 description 7
- 206010033128 Ovarian cancer Diseases 0.000 description 7
- 206010061535 Ovarian neoplasm Diseases 0.000 description 7
- 230000005907 cancer growth Effects 0.000 description 7
- 230000002860 competitive effect Effects 0.000 description 7
- 230000003247 decreasing effect Effects 0.000 description 7
- 239000000539 dimer Substances 0.000 description 7
- 230000005593 dissociations Effects 0.000 description 7
- 229920000669 heparin Polymers 0.000 description 7
- 229960002897 heparin Drugs 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 229910052751 metal Inorganic materials 0.000 description 7
- 239000002184 metal Substances 0.000 description 7
- 238000011580 nude mouse model Methods 0.000 description 7
- 230000000149 penetrating effect Effects 0.000 description 7
- 102220005400 rs34324664 Human genes 0.000 description 7
- 102000009203 Sema domains Human genes 0.000 description 6
- 108050000099 Sema domains Proteins 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 210000002889 endothelial cell Anatomy 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 238000011729 BALB/c nude mouse Methods 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 206010027476 Metastases Diseases 0.000 description 5
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 5
- 206010060862 Prostate cancer Diseases 0.000 description 5
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 5
- 102100027974 Semaphorin-3A Human genes 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 210000004102 animal cell Anatomy 0.000 description 5
- 230000003511 endothelial effect Effects 0.000 description 5
- 210000003527 eukaryotic cell Anatomy 0.000 description 5
- 210000003722 extracellular fluid Anatomy 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 108010082117 matrigel Proteins 0.000 description 5
- 230000009401 metastasis Effects 0.000 description 5
- 229910052755 nonmetal Inorganic materials 0.000 description 5
- PBLQSFOIWOTFNY-UHFFFAOYSA-N 3-methylbut-2-enyl 4-methoxy-8-(3-methylbut-2-enoxy)quinoline-2-carboxylate Chemical compound C1=CC=C2C(OC)=CC(C(=O)OCC=C(C)C)=NC2=C1OCC=C(C)C PBLQSFOIWOTFNY-UHFFFAOYSA-N 0.000 description 4
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 4
- 241000701022 Cytomegalovirus Species 0.000 description 4
- 108010087819 Fc receptors Proteins 0.000 description 4
- 102000009109 Fc receptors Human genes 0.000 description 4
- 101000851030 Homo sapiens Vascular endothelial growth factor receptor 3 Proteins 0.000 description 4
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 102100033179 Vascular endothelial growth factor receptor 3 Human genes 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000004624 confocal microscopy Methods 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-M hexadecanoate Chemical compound CCCCCCCCCCCCCCCC([O-])=O IPCSVZSSVZVIGE-UHFFFAOYSA-M 0.000 description 4
- 239000000710 homodimer Substances 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 208000014018 liver neoplasm Diseases 0.000 description 4
- 239000002082 metal nanoparticle Substances 0.000 description 4
- 238000010232 migration assay Methods 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 210000001236 prokaryotic cell Anatomy 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000003118 sandwich ELISA Methods 0.000 description 4
- 239000004094 surface-active agent Substances 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 239000012739 FreeStyle 293 Expression medium Substances 0.000 description 3
- 108050000637 N-cadherin Proteins 0.000 description 3
- 229920002873 Polyethylenimine Polymers 0.000 description 3
- -1 Sema3F Proteins 0.000 description 3
- 108091008605 VEGF receptors Proteins 0.000 description 3
- 108010065472 Vimentin Proteins 0.000 description 3
- 102100035071 Vimentin Human genes 0.000 description 3
- 230000001745 anti-biotin effect Effects 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 230000007321 biological mechanism Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000008045 co-localization Effects 0.000 description 3
- 239000011258 core-shell material Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000012137 double-staining Methods 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 229960001484 edetic acid Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 206010041823 squamous cell carcinoma Diseases 0.000 description 3
- 238000010254 subcutaneous injection Methods 0.000 description 3
- 239000007929 subcutaneous injection Substances 0.000 description 3
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 230000010474 transient expression Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 210000005048 vimentin Anatomy 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- YHTTWXCDIRTOQX-FQJIPJFPSA-N (6S,9S,15S,18R,23R,26S,29S)-18-amino-6-(4-aminobutyl)-9,26-bis(carboxymethyl)-15-[3-(diaminomethylideneamino)propyl]-2,5,8,11,14,17,25,28-octaoxo-20,21-dithia-1,4,7,10,13,16,24,27-octazabicyclo[27.3.0]dotriacontane-23-carboxylic acid Chemical compound NCCCC[C@@H]1NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]2CCCN2C(=O)CNC1=O)C(O)=O YHTTWXCDIRTOQX-FQJIPJFPSA-N 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 206010011017 Corneal graft rejection Diseases 0.000 description 2
- 241000702421 Dependoparvovirus Species 0.000 description 2
- 206010012689 Diabetic retinopathy Diseases 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 108010021468 Fc gamma receptor IIA Proteins 0.000 description 2
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 2
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 2
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 2
- 102100029193 Low affinity immunoglobulin gamma Fc region receptor III-A Human genes 0.000 description 2
- 101710099301 Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 2
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 108010022871 N-end cysteine peptide tumor-homing peptide Proteins 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- 206010038933 Retinopathy of prematurity Diseases 0.000 description 2
- 102100027751 Semaphorin-3F Human genes 0.000 description 2
- 101710199445 Semaphorin-3F Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 102100034686 Tight junction protein ZO-1 Human genes 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 2
- 208000025865 Ulcer Diseases 0.000 description 2
- 108700007340 Zonula Occludens-1 Proteins 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 210000004082 barrier epithelial cell Anatomy 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 230000009827 complement-dependent cellular cytotoxicity Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000008497 endothelial barrier function Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000004890 epithelial barrier function Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000005732 intercellular adhesion Effects 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 206010023332 keratitis Diseases 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000035168 lymphangiogenesis Effects 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 108010068617 neonatal Fc receptor Proteins 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 231100000956 nontoxicity Toxicity 0.000 description 2
- 238000006384 oligomerization reaction Methods 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 210000003538 post-synaptic density Anatomy 0.000 description 2
- 108010092804 postsynaptic density proteins Proteins 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 231100000397 ulcer Toxicity 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 239000002691 unilamellar liposome Substances 0.000 description 2
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 1
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- OOIBFPKQHULHSQ-UHFFFAOYSA-N (3-hydroxy-1-adamantyl) 2-methylprop-2-enoate Chemical compound C1C(C2)CC3CC2(O)CC1(OC(=O)C(=C)C)C3 OOIBFPKQHULHSQ-UHFFFAOYSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 1
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
- 206010001257 Adenoviral conjunctivitis Diseases 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 206010061424 Anal cancer Diseases 0.000 description 1
- 208000007860 Anus Neoplasms Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 208000009043 Chemical Burns Diseases 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 206010013774 Dry eye Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000001976 Endocrine Gland Neoplasms Diseases 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 241001522878 Escherichia coli B Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 108010001498 Galectin 1 Proteins 0.000 description 1
- 102100021736 Galectin-1 Human genes 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 208000010412 Glaucoma Diseases 0.000 description 1
- 206010018364 Glomerulonephritis Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 208000007514 Herpes zoster Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 1
- 101000851018 Homo sapiens Vascular endothelial growth factor receptor 1 Proteins 0.000 description 1
- 241000193096 Human adenovirus B3 Species 0.000 description 1
- 101100321817 Human parvovirus B19 (strain HV) 7.5K gene Proteins 0.000 description 1
- 108010073807 IgG Receptors Proteins 0.000 description 1
- 102000009490 IgG Receptors Human genes 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 208000002260 Keloid Diseases 0.000 description 1
- 206010023330 Keloid scar Diseases 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000024599 Mooren ulcer Diseases 0.000 description 1
- 101100042271 Mus musculus Sema3b gene Proteins 0.000 description 1
- 101150051337 NRP1 gene Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 208000000821 Parathyroid Neoplasms Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 208000010362 Protozoan Infections Diseases 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 201000002154 Pterygium Diseases 0.000 description 1
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 208000004337 Salivary Gland Neoplasms Diseases 0.000 description 1
- 206010061934 Salivary gland cancer Diseases 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 206010039705 Scleritis Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 208000021386 Sjogren Syndrome Diseases 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 208000021712 Soft tissue sarcoma Diseases 0.000 description 1
- 206010041826 Squamous cell carcinoma of lung Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 208000018656 Terrien marginal degeneration Diseases 0.000 description 1
- 201000007023 Thrombotic Thrombocytopenic Purpura Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 206010064996 Ulcerative keratitis Diseases 0.000 description 1
- 206010046431 Urethral cancer Diseases 0.000 description 1
- 206010046458 Urethral neoplasms Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 1
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 1
- 208000010011 Vitamin A Deficiency Diseases 0.000 description 1
- 208000004354 Vulvar Neoplasms Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 201000005188 adrenal gland cancer Diseases 0.000 description 1
- 208000024447 adrenal gland neoplasm Diseases 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 230000031016 anaphase Effects 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 230000003527 anti-angiogenesis Effects 0.000 description 1
- 230000003302 anti-idiotype Effects 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 201000011165 anus cancer Diseases 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000013602 bacteriophage vector Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Natural products N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000024207 chronic leukemia Diseases 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000013601 cosmid vector Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 208000030381 cutaneous melanoma Diseases 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000009513 drug distribution Methods 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 208000021373 epidemic keratoconjunctivitis Diseases 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical group O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 229940048921 humira Drugs 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 description 1
- 229940099552 hyaluronan Drugs 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000012966 insertion method Methods 0.000 description 1
- 210000002570 interstitial cell Anatomy 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 1
- 210000001117 keloid Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000002197 limbic effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 208000029559 malignant endocrine neoplasm Diseases 0.000 description 1
- 208000026045 malignant tumor of parathyroid gland Diseases 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 210000004088 microvessel Anatomy 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 201000003142 neovascular glaucoma Diseases 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 201000002575 ocular melanoma Diseases 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000004963 pathophysiological condition Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 201000002628 peritoneum cancer Diseases 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000001023 pro-angiogenic effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 238000001403 relative X-ray reflectometry Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 201000004700 rosacea Diseases 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 201000006476 shipyard eye Diseases 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 201000003708 skin melanoma Diseases 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 201000002314 small intestine cancer Diseases 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 201000005102 vulva cancer Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Definitions
- the present invention relates to a tumor tissue-penetrating peptide (TPP) specifically binding to neuropilin.
- TPP tumor tissue-penetrating peptide
- the present invention relates to a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the tumor tissue-penetrating peptide fused therein.
- the present invention relates to a polynucleotide coding the tumor tissue-penetrating peptide, a recombinant vector including the same, a host cell transformed with this vector, and a method for preparing a tumor tissue-penetrating peptide using the host cell.
- the present invention relates to a pharmaceutical composition for treating or preventing cancer, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- the present invention relates to a composition for diagnosing cancer, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- the amount of antibodies transferred to the tumor tissues in actual bodies of human beings is merely 0.01 to 0.001% of the amount injected by various barriers, which means that the treatment effect of antibodies is very limited (Thurber et al. 2008). Accordingly, the development of antibody technology allowing the antibodies to be selectively accumulated in the tumor tissues and to have high permeability into the tumor tissues may increase the treatment effect of antibodies and thus is very important.
- the antibodies for treating the solid tumor are over-expressed in a tumor-associated antigen, or in a tumor, and have a high affinity to a target which is important for the tumor's growth. Even when the antibodies reach the tissues where a specific antigen is present, in the tumor tissues consisting of cells with a great amount of antigen expression, the antibodies are stayed in the antigen while binding to the antigen, due to their high affinity (Lee and Tannock, 2010). Also, after the binding, the antibodies penetrated into the cells along with antigens (Endocytosis) and are lysed. Consequently, the antibodies cannot exert their anti-cancer effects. In order to overcome this, the research for adjusting affinity or lengthening half-life has been processed (Dennis et al. 2007).
- Physiological properties of tumor tissues which prevent the antibodies from being permeated and distributed in the tumor tissues may be broadly classified into 4 cases, which are endothelial barrier, high interstitial fluid pressure, stromal impediment, and epithelial barrier.
- the tumor over-expresses and secretes factors (pro-angiogenic factor) which promote the growth of vascular endothelial cells located around blood vessel in order to receive a great deal of nutrients according to the tumor's rapid speed of growth. Accordingly, a large amount of new blood vessels are produced in an uneven manner, which leads to a decrease in the speed of entire blood flow. In order to overcome this, there is a method for increasing extravasation so that therapeutic agents could come out of the blood vessel and be distributed into the tissue.
- TNF- ⁇ and IL02 which are cytokine inflammatory responses related with extravasation, chemical substance promoting extravasation (promoter chemical drug) (Marcucci et al. 2013), iRGD peptide, etc. in combination with therapeutic agents.
- promoter chemical drug promoter chemical drug
- iRGD peptide reached its limit in that it needs to be administered in a great quantity (2 mg/kg or 4 mg/kg) (Sugahara et al. 2010).
- High tumor interstitial fluid pressure results from a situation where the pressure difference allowing the drug to be convected from the blood vessel to the tissue is small, or where the fluid pressure of tissue is higher than that of blood.
- High tumor interstitial fluid pressure is mainly caused due to the accumulation of interstitial fluid pressure in the absence of lymphatic duct in the tumor tissue, unlike in the normal tissue, and contributes to abnormal angiogenesis.
- a method for preventing the operation of a factor promoting the growth of vascular endothelial cell, particularly vascular endothelial cell growth factor-A (VEGF-A), and inhibiting angiogenesis to normalize the blood vessel, or a method for increasing the fluid pressure of blood vessel has been attempted.
- VEGF-A vascular endothelial cell growth factor-A
- the stromal impediment which is an extracellular matrix barrier met when the antibodies come out to micro-vessels and are convected to the tissue, mainly consists of collagen and hyaluronan.
- the extracellular matrix greatly affects the shape of tumor. Accordingly, there is a big difference between the area where the drug is well distributed and the area where the drug is not well distributed, so drug distribution becomes uneven. Additionally, as an amount of expression of extracellular matrix increases, the tumor interstitial fluid pressure due to a high cell density with solid tumor stress (solid stress) increases. As the method for overcoming this, there is a method for inducing apoptosis of tumor tissue cell to reduce tumor interstitial cell density. Additionally, there was a case increasing the drug delivery effect about two times compared to a control group by processing an enzyme (collagenase) dissolving collagen in the tumor tissue to reduce solid stress (Eikenes et al. 2004).
- E-cadherin is well known as a main factor of the intercellular adhesion. Since a substance reducing the E-cadherin was found in virus (adenovirus-3), there was a case where only a part (JO-1) with an activity of reducing E-cadherin of cell among proteins constituting the virus was administered in combination with the antibody, thereby increasing an anti-cancer effect of antibody (Beyer et al. 2011).
- peptide is pharmacokinetics resulting from a small size of molecule, and has very short half-life.
- a great amount of peptide needs to be administered to actual patients and the administration needs to be frequently made.
- the therapeutic agents and substances for tumor permeation operation need to be produced, respectively, which is an inevitable process during co-administration, its industrial practicability is low.
- the peptide sequence and protein which do not exist in the natural world are likely to cause immunogenicity.
- it is required to develop a format where the antibody acquires tissue permeability as it is so that the delivery effect of one antibody molecule into the tumor tissue could be increased.
- VEGF-A vascular endothelial cell growth factor-A
- extravasation also called as a vascular permeability factor.
- VEGFR2 vascular endothelial cell growth factor receptor
- Neuropilin was first found in a Xenopus nervous system. Neuropilin is a transmembrane glycoprotein, and has two types of NRP1 and NRP2. Neuropilin is expressed very weakly in normal cells, whereas is over-expressed in tumor vascular endothelial cells, solid tumor cells, blood tumor cells. Neuropilin operates as a coreceptor of VEGFRs (VEGF receptors) by binding to VEGF family ligands. Especially, NRP1 operates as a coreceptor of VEGFR1, VEGFR2 and VEGFR3, and binds to various VEGF ligands, thereby contributing to angiogenesis, cell survival, migration & adhesion, invasion, etc. in the tumor tissue.
- VEGFRs VEGF receptors
- NRP2 operates as a coreceptor of VEGFR2 and VEGFR3, thereby contributing to lymphangiogenesis and cell adhesion.
- NRP1/NRP2 (NRP1/2) operates as a coreceptor of plexin family receptors to bind to secreted class 3 semaphorins (Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, Sema3F and Sema3G). Since the neuropilin has no domain in functional cells, even if a ligand is binding thereto, the neuropilin has no activity by itself.
- VEGF receptor which is the coreceptor, or through the plexin co-receptor.
- Sema3 binds to neuropilin and the plexin receptor at a ratio of 2:2:2 and operates.
- anti-neuropilin-2 antibody In the case of anti-neuropilin-2 antibody, it is reported that anti-neuropilin-2 antibody is competitive to bind to neuropilin-2 with VEGA-C which is well known for binding to VEGFR3 and neuropilin-2 at the same time, and has the function of inhibiting lymphangiogenesis and cell adhesion, which are operations of VEGFR3 (Cant M et al. 2008).
- the present inventors infer a part of minimum Sema3A- or Sema3F-derived peptide with an enhancing effect of vascular endothelial cell permeability by the interaction between the neuropilin and Sema3A or Sema3F, and designed a mutant peptide so as to have a high affinity with the neuropilin.
- the present inventors designed bivalent shape by converging peptide into a heavy-chain C-terminus of antibody so that the peptide could copy the function of Sema3A/Sema3F operating as a homodimer. As this design is a single molecule fused with antibody-peptide, a unique function of antibody is maintained as it is.
- a fusion antibody technology with an anti-angiogenesis effect has been developed.
- TPP tumor tissue-penetrating peptide
- a pharmaceutical composition for treating or preventing cancer or angiogenesis-related diseases including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- compositions for diagnosing cancer or angiogenesis-related diseases including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- An aspect of the present invention provides a tumor tissue-penetrating peptide (TPP) specifically binding to neuropilin.
- TPP tumor tissue-penetrating peptide
- Another aspect of the present invention provides a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the tumor tissue-penetrating peptide fused therein.
- Another aspect of the present invention provides a polynucleotide coding the tumor tissue-penetrating peptide, a recombinant vector including the same, a host cell transformed with this vector, and a method for preparing a tumor tissue-penetrating peptide using the host cell.
- another aspect of the present invention provides a pharmaceutical composition for treating or preventing cancer or angiogenesis-related diseases, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- another aspect of the present invention provides a composition for diagnosing cancer or angiogenesis-related diseases, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- the tumor tissue-penetrating peptide and a protein having the peptide fused therein of the present invention have the properties of specifically binding to neuropilin, and accordingly specifically accumulate in tumor tissues, widen intercellular gap between the tumor vascular endothelial cells to promote extravasation, and control intercellular gap between tumors within the tumor tissue to increase infiltration within tumor tissue to show a remarkably increased in vivo tumor-suppressing activity.
- a fusion antibody having Sema3A- and Sema3F-derived peptides and peptides with remarkably improved affinity of their wild-type peptides with neuropilin fused therein or in the antibody fragment are administered in the same dosage as a control group antibody having no peptide fused therein, they specifically accumulate in tumor tissues, and increase infiltration within tumor tissue, to show a remarkably increased in vivo tumor-suppressing activity, compared with the control group antibody.
- An antibody having the tumor tissue-penetrating peptide of the present invention fused therein or a fragment thereof has the property of a bispecific antibody which has a binding capacity with neuropilin to which a tumor tissue-penetrating peptide binds, while maintaining the antigen binding capacity which the antibody originally has, and accordingly accumulates a fusion antibody with high efficiency in tumor tissues and increases infiltration within tumor tissues and thus is expected to have a high effect in treating and diagnosing tumor.
- a fragment of the antibody having the tumor tissue-penetrating peptide of the present invention fused therein has the property of inhibiting angiogenesis by VEFG165A by inhibiting VEFG165A from binding to neuropilin 1/2, and thus is expected to be used for treating and diagnosing various diseases such as diabetic retinopathy, rheumatoid arthritis, or atherosclerosis, relating to angiogenesis.
- an antibody having the peptide of the present invention fused therein or a fragment thereof shows a production yield similar to wild-type antibody which does not have a peptide fused therein, and thus there is no problem in mass production.
- the fusion antibody or a fragment thereof maintains the antigen binding capacity which the wild-type antibody originally has, unique function of heavy-chain constant region (Fc), i.e., binding to FcRn (neonatal Fc receptor), and accordingly has a long serum half-life. Also, it has an advantage that the binding site (protein A and protein G) is preserved during the purification process, and the antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity may be maintained.
- FIG. 1 is a schematic diagram illustrating the structures of semaphorins, VEGF165A, and neuropilin-1 and -2.
- semaphorin broadly includes three domains. From the N-terminus, a sema domain is a site binding to plexin, an Ig-like C2 type domain is a site binding to neuropilin a1 and a2, and a basic rich region is a site exposing a portion capable of binding to neuropilin by being cleaved by furin.
- VEGF165A includes a site capable of binding to VEGFR2 (KRD) which is an original receptor of VEGF and a heparin-binding domain (HBD) capable of binding to neuropilin. Further, in each of semaphorin and VEGF165A, an interface forming a dimer is present.
- KRD VEGFR2
- HBD heparin-binding domain
- neuropilin broadly consists of five domains, and from the N-terminus, a1 and a2 domains are classified as CUB domains, and an Ig-like C2 type domain of semaphorin binds thereto. Particularly, this site forms a complex with plexin to increase binding capacity with semaphorin-plexin.
- the b1 and b2 domains are classified as FV/VIII domains, and the C-terminus of ligands of VEGF or class 3 semaphorins bind thereto. Particularly, in this portion, a site to which heparin is capable of binding is present and this facilitates the binding of ligands with many (+) charged residues.
- trans-membrane domain enables neuropilin to be fixed onto cell surface, and in a cytosolic domain, a site capable of binding to a Postsynaptic density 95, Disk large, Zona occludens 1 (PDZ) domain is present.
- FIG. 2 is a schematic diagram illustrating a 2:2:2 complex including plexin of Sema3A and its co-receptor neuropilin.
- Sema3A and Sema3F present in nature form a homodimer, and the Sema domain interacts with the Sema domain of plexin. Further, the C-terminus of semaphorin interacts with b1 domain of neuropilin.
- FIG. 3 is a schematic diagram illustrating a binding complex of neuropilin, ligand, and TPP fused antibody heavy-chain constant region (Fc-TPP). It was expected that each protein at its C-terminus binds to neuropilin (particularly, to b1 domain) in the form of dimer.
- Fc-TPP fused antibody heavy-chain constant region
- FIG. 4 is a schematic diagram illustrating a vector for expressing Fc-TPP in animal cells.
- FIG. 4(A) illustrates a part encoding Fc-TPP of a cleavage map of a vector for expression in host cells.
- FIG. 4(B) illustrates a whole map of a vector for expressing Fc-TPP in host cells.
- FIG. 5 is a schematic diagram illustrating TPP fused antibody heavy-chain constant region, and expression and purification SDS-PAGE.
- the antibody heavy-chain constant region was prepared starting from a hinge at N-terminus to maintain two disulfide bonds and facilitate formation of a dimer.
- the external exposure degree of TPP was regulated using the peptide linker of four amino acids GAGA or fifteen amino acids (G4S)3 at the end of CH3, and thereafter 22 semaphorin derived sequences and improved derived sequences were added for preparation.
- FIG. 6 illustrates a result of ELISA experiment for confirming the binding to b1b2 domains of NRP-1 and -2.
- VEGFR2-Fc, and b1b2 domains of NRP-1 and -2 were fixed onto plates, biotinylated peptides and Fc-TPP clones were bound at a concentration of 100 nM (in the case of peptides, 100 nM, 1 ⁇ M), and the binding was confirmed using anti-biotin antibody-alkaline phosphatase (AP) (SIGMA-ALDRICH co., USA) and pnitrophenyl palmitate (pNPP, SIGMA-ALDRICH co., USA).
- AP anti-biotin antibody-alkaline phosphatase
- pNPP SIGMA-ALDRICH co., USA
- FIG. 6(A) illustrates a comparison of the control group with Sema3A derived peptide and Fc-TPP
- FIG. 6(B) illustrates a comparison of the control group with Sema3F derived peptide and Fc-TPP.
- stronger bond to neuropilin1/2 occurred in A22p and F22p peptides and Fc-A22p/F22p clones inducing mutants, and slightly stronger bond occurred in clones using (G4S)3 linker among clones using GAGA and (G4S)3 linkers.
- No bond to the control group VEGFR2 was observed. Thereby, it was shown that TPP selectively binds to neuropilin.
- FIG. 7 confirms binding specificity of clones showing stronger binding capacity to neuropilin among Fc-TPP after confirming whether these clones are competitive with VEGF165A or Semaphorin3A to bind to neuropilin. It was confirmed that Fc-15A22p is competitive at a higher concentration of ligand than Fc-15F22p.
- FIG. 8 illustrates a result of FACS analysis of expression level of neuropilin-1 and -2 on the surface of cell lines used for biological identification of TPP in this experiment.
- PPC-1 human prostatic cancer cell line
- FaDu human head and neck cancer cell line
- HAVEC human endothelial cell line
- SK-OV-3 human ovarian cancer cell line
- FIG. 9(A) illustrates a result of FACS analysis in human ovarian cancer cell line (SK-OV-3) in order to confirm whether Fc-TPP binds to neuropilin-1 and -2 expressed on the cell surface.
- Fc, Fc-15A22P, and Fc-15F22P having binding capacity to b1b2 domains of neuropilin-1 and -2 were treated under the same conditions to confirm their binding capacities.
- FIG. 9(B) is a view illustrating a result confirming the binding capacity using the same manner as in the experiment of FIG.
- FIGS. 9(C) and 9(D) illustrates a result of FACS analysis for comparing binding sites among VEGF165A, Sema3A, and Sema3F, which have binding capacity to neuropilin-1 and -2.
- VEGF165A the binding capacity of Fc-TPP was inhibited by VEGF165
- the binding capacity of Fc-15A22P by Sema3A the binding capacity of Fc-15F22P by Sema3F.
- FIG. 10 illustrates a result of observation of co-localization of Fc-TPP and neuropilin-1 and -2 through confocal microscopy analysis, in order to confirm the specific intracellular penetrating capacity of Fc-TPP into neuropilin.
- FIG. 11 a illustrates a result of Western blot for identifying biological mechanism of Fc-TPP in HUVEC.
- single forms of peptide, A22, A22P, F22, and F22P were used, and for comparison depending on linker length with TPP, fusion types of Fc format, Fc-4A22, Fc-15A22, Fc-15A22P, Fc-4F22, Fc-15F22, and Fc-15F22P were used.
- the control group VEGF165A and Sema3A unlike Sema3F, showed improvement of permeability in HUVEC, and this can be indirectly confirmed by a decrease in VE-cadherin.
- FIG. 11 b illustrates a result of Transwell assay performed for confirming whether TPP improves permeable capacity of HUVEC.
- VEGF165, Sema3A, Fc-15A22, Fc-15A22P, and Fc-15F22P effectively improved permeability.
- single forms of peptide, A22, A22P, F22, and F22P did not improve permeability. This result is closely related to the result of FIG. 11 a.
- FIG. 11 c illustrates a result of immunohistochemistry for confirming TPP's infiltration capacity in actual tumor tissues.
- Human epidermoid carcinoma cell line A431 was transplanted into nude mice to confirm the effect of TPP through double staining with blood vessels (CD31). As a result, it was confirmed that unlike peptide A22P and the control group Fc, Fc-15A22 and Fc-15A22P selectively reached tumor cells, and effectively infiltrated into tumor cells.
- FIG. 11 d illustrates a result of confirming the improvement of vascular permeability through Evans Blue assay. As a result, it was confirmed that unlike Fc, Fc-15A22P effectively improved tissue infiltration capacity, as shown in the above result.
- FIG. 11 e illustrates a result of Western blot for a change in E-cadherin in human head and neck cancer cell line FaDu, in order to confirm the effect in cancer cell as well as vascular endothelial cell.
- Fc-15A22 and Fc-15A22P induced a decrease in E-cadherin, unlike Sema3A, and in the case of Sema3F derived TPP, Fc-15F22P, like Sema3F, induced a decrease in E-cadherin.
- Fc-TPP did not cause a decrease in E-cadherin, and thereby it was confirmed that Fc-TPP induced a decrease in E-cadherin specifically to neuropilin.
- FIG. 12 a illustrates a result of tube formation assay performed for confirming whether Fc-15A22P inhibits tube formation of vascular endothelial cell by VEGF165A. As a result, it was confirmed that Fc-15A22P effectively inhibited the tube formation of endothelial cell induced by VEGF165A.
- FIG. 12 b performed in vivo matrigel plug assay on mice, in order to identify whether Fc-15A22P can inhibit angiogenesis induced by VEGF165A. Also, the density of blood vessels was measured with anti-CD31 antibody through immunohistochemistry. As a result, it was confirmed that Fc-15A22P can inhibit angiogenesis induced by VEGF165A.
- FIG. 13(A) is a schematic diagram illustrating a fusion antibody (mAb-TPP) where a peptide binding to neuropilin is linked by a peptide linker to heavy-chain constant region C-terminus of single clone antibody.
- mAb-TPP fusion antibody
- FIG. 13(B) is a schematic diagram illustrating that the fusion antibody of (A) binds to b1 domain of neuropilin-1 or -2.
- FIG. 13(C) is a schematic diagram illustrating the mechanism expected when the above fusion antibody (mAb-TPP) is introduced into the body.
- the binding of peptide fused antibody to neuropilin-1 or -2 overexpressed in tumor vascular endothelium and various tumor cells increases selective distribution of tumor tissue, increases extravasation, and induces signal transduction increasing infiltration into tumor cells, which may result in remarkably increasing specific distribution of tumor tissue of fusion antibody and infiltration thereinto.
- FIG. 14 is an example of a cleavage map of a vector for expressing IgG heavy chain-TPP.
- FIG. 14(A) represents a part coding IgG heavy chain-TPP of map of a vector for expression in host cells.
- FIG. 14(B) represents a whole map of a vector for expressing IgG heavy chain-TPP in host cells.
- FIG. 15 is an example of a cleavage map of a vector for expressing IgG light chain in animal cells.
- FIG. 15(A) represents a part coding IgG light chain of map of a vector for expression in host cells.
- FIG. 15(B) represents a whole map of a vector for expressing IgG light chain in host cells.
- FIG. 16(A) is a schematic diagram illustrating an antibody constructed by introducing TPP into Fc C-terminus of Cetuximab IgG which is the existing anti-EGFR IgG.
- FIG. 16(B) illustrates a result of analyzing the size and purity on SDS-PAGE under reducing and non-reducing conditions, after transient expression and purification in HEK293K cells through co-transformation.
- FIG. 16(C) illustrates a result confirming through Sandwich ELISA that Cetuximab-TPP is capable of binding to antigen EGFR and neuropilin simultaneously.
- FIG. 17(A) is a schematic diagram illustrating an antibody introducing TPP into the existing anti-HER2 IgG Trastuzumab in the same manner as the experiment above.
- FIG. 17(B) illustrates a result of analyzing the size and purity on SDS-PAGE under reducing and non-reducing conditions, after transient expression and purification in HEK293F cells through co-transformation.
- FIG. 17(C) illustrates a result confirming through Sandwich ELISA that Trastuzumab-TPP is capable of binding to antigen Her2 and neuropilin simultaneously.
- FIG. 18 illustrates a result of Surface Plasmon Resonance (SPR) for confirming whether binding capacity between mAb and mAb-TPP depending on pH for FcRn is similar to each other. It was proven that mAb (Cetuximab, Trastuzumab) showed a binding curve at pH 6.0, and mAb-TPP (Cetuximab-TPP, Trastuzumab-TPP) maintained the properties of wild-type antibodies which do not completely bind at pH 7.4.
- SPR Surface Plasmon Resonance
- FIG. 19 a illustrates a result of IHC for confirming the infiltration capacity of Cetuximab-TPP in tumor tissues.
- FIG. 19 b illustrates a result of Western blot performed from cancer cell tissues extracted from mice under the conditions of FIG. 18 b.
- FIG. 19 c illustrates a result of experiment on inhibition of cancer cell growth in nude mice for confirming whether improved infiltration into tumor tissue actually affects inhibition of cancer cell.
- Cetuximab and Cetuximab-15A22P inhibited the growth of cancer cell, compared with PBS, and that Cetuximab-15A22P more effectively inhibited the growth of cancer cell under the same conditions.
- FIG. 19 d illustrates a result of measuring the weights of mice at the time of conducting the above experiment.
- the experimental group into which Cetuximab was injected had no difference in weight. This indirectly proves that Cetuximab-15A22P does not have any toxicity against mice, compared with Cetuximab.
- FIG. 20 a illustrates a result of IHC for confirming the infiltration capacity of Trastuzumab-TPP in tumor tissues.
- FIG. 20 b illustrates a result of Western blot performed from cancer cell tissues extracted from mice under the conditions of FIG. 19 a.
- FIG. 20 c illustrates a result of experiments for confirming the effect of Trastuzumab-TPP on inhibiting the growth of cancer cell. As a result, it was confirmed that under the same conditions, Trastuzumab-15A22P more effectively induced inhibition on the growth of cancer cell than Trastuzumab.
- FIG. 20 d illustrates a result of measuring weights of mice during the above experiments.
- mice of the experimental group into which Trastuzumab was injected had no difference in weights. This indirectly proved that Trastuzumab-15A22P did not have any toxicity against mice, when compared with Trastuzumab.
- FIG. 21 illustrates a result of in vitro evaluation on the degree of cell growth inhibition by treating human head and neck cancer cell line FaDu and human ovarian cancer cell line SK-OV-3 with Fc-TPP and mAb-TPP.
- Cetuximab and Cetuximab-15A22P inhibited cell growth by about 30% in cell line FaDu
- Trastuzumab and Trastuzumab-15A22P inhibited cell growth by about 30-35% in cell line SK-OV-3.
- Fc and Fc-TPP did not show an effect of inhibiting cell growth in both FaDu and SK-OV-3.
- the mechanism of TPP by neuropilin means that TPP itself maximizes the effect by increasing infiltration into tumor tissue without directly affecting the growth of cancer cells.
- VEGF-A vascular endothelial cell growth factor
- B, C, D semaphorin 3-A
- B, C, D, E class 3 semaphorin
- FGF2 fibroblast growth factor-2
- HGF hepatocyte growth factor
- VEGF-A and class 3 semaphorin were selected from the PubMed Entrez Protein Database.
- secreted class 3 semaphorins (Sema3s) were identified from semaphorins 3A to 3G, and they have three domains in common.
- a sema domain is a site binding to plexin
- an Ig-like C2 type domain is a site binding to neuropilin a1 and a2
- a basic rich region is a site exposing a portion capable of binding to neuropilin by being cleaved by furin.
- VEGF-A which most strongly binds to neuropilin, among ligands of neuropilin known until now, particularly, VEGF165A, is divided into a site capable of binding to a primary receptor of VEGF, VEGFR2 and a heparin-binding domain (HBD) capable of binding to neuropilin.
- HBD heparin-binding domain
- VEGF165A contains a portion (115-159, Exon7a, 7b) where there are many (+) charged residues binding to heparin which binds to neuropilin-1, in addition to the portion (160-165, Exon8a) binding to neuropilin-1.
- VEGF165A contains cysteine at a portion corresponding to Exon8, and thus is expected to cause a decrease in an amount of expression, when VEGF165A is fused with a protein such as an antibody and expressed, and thus it is excluded.
- neuropilin broadly consists of five domains, and from the N-terminus, a1 and a2 domains are classified as CUB domains, and an Ig-like C2 type of semaphorin binds thereto. Particularly, this site forms a complex with plexin, and plays a role of increasing the binding force with semaphorin-plexin.
- the b1 and b2 domains are classified as FV/VIII domains, and the C-terminus of VEGF family ligand or class 3 semaphorin ligand binds thereto.
- a site to which heparin is capable of binding is present and this facilitates the binding of ligands with many (+) charged residues.
- MAM induces oligomerization, trans-membrane domain (TM) enables neuropilin to be fixed onto cell surface, and in a cytosolic domain, a site capable of binding to a Postsynaptic density 95, Disk large, Zona occludens 1 (PDZ) domain is present.
- TM trans-membrane domain
- FIG. 2 is a schematic diagram illustrating a 2:2:2 complex including plexin of Sema3A and its co-receptor neuropilin.
- Sema3A and Sema3F present in nature form a homodimer, and the Sema domain interacts with the Sema domain of plexin. Further, the C-terminus of semaphorin interacts with b1 domain of neuropilin.
- FIG. 3 is a schematic diagram illustrating a binding complex of neuropilin, ligand, and TPP fused antibody heavy-chain constant region (Fc-TPP). It was expected that similar to Sema3A and Sema3F present in nature, each protein at its C-terminus binds to neuropilin (particularly, to b1 domain) in the form of dimer.
- Fc-TPP fused antibody heavy-chain constant region
- Sema3A CEQVWKRDRKQRRQRPGHTFGNSNKWKHLQENKKGR N RR (722-760) Sema3B CRP-----QPALQSL--PLE----------SRRKGR N RR (710-731) Sema3C CKDTRQQRQQGDESQ--KMRGDYGKLKALINSRKSR N RR (709-745) Sema3D CEQMWRREK--RRQR---NKGGP-KWKRMQEMKKKR N RR (731-763) Sema3E CTDRKRKKLKMSPSK--WKYANPQEKKLRSKPERYR L PR (734-770) Sema3F CQGYWRHVPPSPREA----PGAP-RSPEPQDQKKPR N RR (756-779) Sema3G CFRSRSRGKQARGKS----------WA---GLELGK K MK (750-774) VEGF165 CK
- 22 amino acids were selected from the C-terminus sequence information of each of Sema3A and Sema3F of which binding capacity to neuropilin is well known. Specifically, A22 peptide having 22 amino acids derived from residues 739-760 which are part of the basic domain of Sema3A and F22 peptide having 22 amino acids derived from residues 758-779 which are part of the basic domain of Sema3F were selected.
- A22p and F22p where the last third amino acids of A22 and F22 are replaced with proline were designed.
- the following Table 3 shows peptide sequences and SEQ ID NO. of A22 and F22 having amino acid sequences derived from C-terminus of Sema3A and Sema3F binding to neuropilin, and of A22p and F22p designed to increase affinity with neuropilin.
- the underlines are used to highlight the parts inducing mutants.
- TPP Tumor Tissue-Penetrating Peptide
- linkers having 4 amino acids consisting of glycine, serine, and alanine, or 15 amino acids were selected.
- the selected linkers have sequences of GAGA and (GGGGS)3.
- the clone names and sequence information are shown in the following Table 4.
- Peptides (SEQ ID NOS: 5-10) shown in Table 4 were fused to C-terminus of Fc, a constant region of human antibody IgG1, to have the properties of binding neuropilin in a bivalent form. This is designed to activate target neuropilin receptors, by copying Sema3A and Sema3F ligands which bind to neuropilin as homodimer.
- FIG. 4(A) illustrates a part coding Fc-TPP of a cleavage map of a vector for expression in host cells.
- FIG. 4(B) illustrates a whole map of a vector for expressing Fc-TPP in host cells.
- Fc was cloned into the pSecTag2A vector with the restriction enzyme AscI/HindIII.
- Each Fc-TPP was substituted with the restriction enzyme BsrGI/HindIII from CH3 till the terminus by performing PCR with same forward primers starting from CH3 having Fc as a template and their respective reverse primers for introducing TPP into C-terminus.
- Fc and Fc-TPP show different sizes as much as those of introducing TPP to purified proteins on SDS-PAGE ( FIG. 5(B) )
- Fc was used as a control group for the size.
- Biotin-peptide of sequence derived from semaphorin consisting of 22 amino acids which was used as a control group was prepared by chemical synthesis (Peptron, Korea).
- Proteins were expressed using transient transfection of plasmid ( FIG. 4 ) encoding each Fc-TPP constructed in Example 2 above, with HEK293-F system (Invitrogen).
- HEK293-F cells Invitrogen
- serum-free FreeStyle 293 expression medium Invitrogen
- PEI polyethylenimine
- plasmid encoding Fc-TPP was diluted in 10 ml FreeStyle 293 expression medium (Invitrogen) to be 250 ⁇ g (2.5 ⁇ g/ml), and mixed with 10 ml medium where PEI 750 ⁇ g (7.5 ⁇ g/ml) was diluted to be reacted at room temperature for 10 minutes. Then, the reacted mixture medium was put into the 100 ml seeded cells and incubated at 120 rpm, 8% CO 2 for 4 hours, and then the other 100 ml FreeStyle 293 expression medium was added thereto, followed by incubation for 7 days. The supernatant was collected after 7 days.
- 10 ml FreeStyle 293 expression medium Invitrogen
- Proteins were purified from the collected cell culture supernatant by referring to the standard protocol. Antibodies applied to Protein A Sepharose column (GE healthcare) and were washed with PBS (pH 7.4). The antibodies were eluted at pH 3.0 using 0.1 M glycine buffer, and thereafter samples were immediately neutralized using 1 M Tris buffer. The buffer was changed to PBS (pH 7.4) using Pierce Dextran Desalting Column (5K MWCO). Thereafter, the eluted antibody fragments were concentrated using the centrifugal concentrator MILLIPORE Amicon Ultra (10 MWCO), and purified Fc-TPP was quantified using absorbance and absorption coefficient at wavelength 280 nm. The purified Fc-TPP was analyzed on SDS-PAGE under reducing and non-reducing conditions.
- FIG. 5(A) is a schematic diagram illustrating TPP fused antibody heavy chain constant region.
- the antibody heavy-chain constant region was prepared starting from a hinge at the N-terminus to maintain two disulfide bonds and facilitate formation of a dimer.
- the external exposure degree of TPP was regulated using the peptide linker of GAGA or (GGGGS)3 at the end of CH3, and thereafter 22 semaphorin derived sequences were added for preparation.
- FIG. 5(B) illustrates a result of analysis of SDS-PAGE of the purified Fc-TPP under reducing and non-reducing conditions. From FIG. 5(B) , the formation and purity of dimmers of each clone on SDS-PAGE can be confirmed.
- Table 5 shows the yields of proteins produced per 1 L medium of the purified TPP fused protein. The results obtained from three times experiments were statistics processed, and ⁇ represents standard deviation. The obtained yields of proteins are not remarkably different from those of wild-type proteins.
- VEGF165A, Sema3A (26-760), Sema3F (19-779) and Fc, and their respective Fc-TPPs were biotinylated using the NHS-biotin kit (SIGMA-ALDRICH co., USA).
- the target molecules, b1b2 domains (273-586) of neuropilin-1 and b1b2 domains (275-595) of neuropilin-2, and the control group VEGFR2 (46-753) were bound in an amount of 1 ⁇ g each in 96-well EIA/RIA plates (COSTAR Corning In., USA) at room temperature for 1 hour, and then washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA).
- biotinylated VEGF165A, Sema3A, Sema3F and Fc were washed three times for 10 minutes with 100 nM (or were bound in a concentration of 1 ⁇ M of peptide and thereafter washed three times for 10 minutes with 0.1% PBST).
- AP alkaline phosphatase
- pNPP pnitrophenyl palmitate
- absorbance at 405 nm was quantified. From the ELISA result obtained from 30 minute-reaction of AP-pNPP, the binding capacity of expressed and purified Fc-TPP to b1b2 domains of neuropilin-1 and -2 was confirmed.
- FIG. 6 illustrates a result of ELISA experiments for confirming the binding to b1b2 domains of neuropilin-1 and -2.
- FIG. 6(A) illustrates a comparison of the control group with Sema3A derived peptide and Fc-TPP
- FIG. 6(B) illustrates a comparison of the control group with Sema3F derived peptide and Fc-TPP.
- b1b2 domains (273-586) of neuropilin-1 and b1b2 domains (275-595) of neuropilin-2 were bound in 96-well EIA/RIA plates (COSTAR Corning In., USA) at room temperature for 1 hour, and then washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA).
- FIG. 7 illustrates a result of confirmation whether clones showing stronger binding force to neuropilin among Fc-TPP are competitive with VEGF165A and Sema3A to bind to neuropilin. As illustrated in FIG. 7 , it was confirmed that Fc-15A22p is competitive at a higher concentration of ligand than Fc-15F22p.
- each b1b2 domain of neuropilin-1 and -2 was diluted with 10 mM Na-acetate buffer (pH 4.0), and fixed onto CM5 sensor chips (GE healthcare, USA) in about 1000 response units (RU).
- HBS-EP buffer (10 mM Hepes, 3 mM ethylenediaminetetraacetic acid, and 0.005% surfactant P20 (pH 7.4), GE Healthcare) was analyzed at a stream velocity 30 ⁇ l/min, and VEGF165 at a concentration of 80 nM to 5 nM, Sema3F and Sema3A at a concentration of 1 ⁇ M to 62.5 nM, and Fc-TPP at a concentration of 25 ⁇ M to 1.5625 ⁇ M were analyzed.
- CM5 chip After bond and dissociation analysis, the regeneration of CM5 chip was performed by streaming a buffer (20 Mm NaOH, 1 M NaCl, pH 10.0) at a stream velocity 30 ⁇ l/min for 1 minute. Each sensorgram obtained by 3 minute-bond and 3 minute-dissociation was normalized and subtracted by comparing with blank cells to calculate the binding affinity.
- Table 6 shows a result of the binding affinity of Sema3A derived A22 peptide and a single mutant A22p in the form of Fc-TPP with b1b2 domains of neuropilin-1 and -2 (NRP 1 and 2), using surface plasmon resonance (SPR, BIACORE 2000, GE healthcare, USA).
- Table 7 shows a result of the binding affinity of Sema3F derived F22 peptide and a single mutant F22p in the form of Fc-TPP with b1b2 domains of neuropilin-1 and -2.
- the difference in binding affinity between clones having GAGA linker and clones having (GGGGS)3 linker is about 10 times, when compared with Fc-4A22 and Fc-15A22.
- Pro mutant clones show the binding affinity about 100 times as high as those fused with wild-type peptide binding to neuropilin. At least 5 sensorgrams were used for analysis, and results obtained from twice experiments were statistics processed. ⁇ represents standard deviation of results of individual experiments.
- FIG. 8 is a view illustrating that neuropilin-1 and -2 are expressed in cell lines.
- PPC-1 human prostatic cancer cell line
- FaDu human head and neck cancer cell line
- HAVEC human endothelial cell line
- SK-OV-3 human ovarian cancer cell line
- Fc-TPP binds to neuropilin-1 and -2 expressed on cell surface
- FACS analysis was performed, and Fc-15A22P and Fc-15F22P which were confirmed to specifically bind to neuropilin-1 and -2 were used.
- FIG. 9 illustrates a result of FACS analysis in human prostate cancer cell line (PPC-1) in order to confirm whether Fc-TPP binds to neuropilin-1 and -2 expressed on the cell surface. As illustrated in FIG. 9(A) , it was confirmed that unlike Fc, Fc-15A22P and Fc-15F22P bind to the surface of PPC-1 cells.
- each of Fc-15A22P and Fc-15F22P was mixed with VEGF165A (1 ⁇ g/ml), and the binding capacity was confirmed in the same manner as above. As illustrated in FIG. 9(C) , it was confirmed that the binding capacity was reduced by VEGF165A, and this means competitive binding with VEGF165A.
- Fc-15A22P and Fc-15F22P were mixed with Sema3A and Sema3F (100 ⁇ g/ml), respectively, which are ligands from which Fc-15A22P and Fc-15F22P were derived, and reacted, to confirm the binding capacity.
- Sema3A and Sema3F 100 ⁇ g/ml
- the binding capacity of each of Fc-15A22P and Fc-15F22P was reduced by Sema3A and Sema3F. This means that the binding of Fc-TPP to neuropilin-1 and -2 is specific similar to the original ligands.
- Fc-TPP has intracellular penetrating capacity by neuropilin-1 and -2, like other neuropilin ligands having intracellular penetrating capacity, confocal microscopy was used to observe intracellular penetration and co-localization with neuropilin-1 and -2.
- PPC-1 cells were plated into 24-well plates containing DMEM medium 0.5 ml supplemented with 10% FBS per well, followed by incubation at 37° C. for 24 hours at 5% CO 2 .
- DMEM medium 0.5 ml supplemented with 10% FBS per well
- Fc, Fc-15A22P, and Fc-15F22P were diluted with 0.5 ml of transfection optimized medium (TOM, WelGENE Inc., Korea) to be 1 ⁇ M, followed by incubation at 37° C. for 1 hour under 5% CO 2 condition.
- TOM transfection optimized medium
- Fc-TPP was stained with an antibody (Sigma) which specifically recognizes the FITC (green fluorescence)-conjugated Fc, and neuropilin-1 and -2 was stained with a primary antibody (SantaCruz) which recognizes each of them and with a TRITC (red fluorescence)-conjugated secondary antibody (Sigma). Nuclei were stained (blue fluorescence) with DAPI and observed under confocal microscopy.
- FIG. 10 illustrates a result of observation of co-localization of Fc-TPP and neuropilin-1 and -2 through confocal microscopy analysis, in order to confirm the specific intracellular penetrating capacity of Fc-TPP through neuropilin.
- FIG. 10 it was confirmed that the control group Fc did not penetrate into cells, whereas Fc-15A22P and Fc-15F22P penetrated into cells and were co-localized with neuropilin-1 and -2, respectively. This means that Fc-TPP has specific penetrating capacity by neuropilin-1 and -2.
- Sema3A or VEGF165A is known to increase vascular permeability using neuropilin-1 (NRP1) as a co-receptor.
- NBP1 neuropilin-1
- VE-cadherin or epithelial (E)-cadherin involves in adhesion between endothelial cells, while forming the base of adherent junction between endothelial cells and epithelial cells. The change in molecules relieve the density of adherent junction, which results in increasing permeability of blood vessels and improving intracellular permeability.
- VE-cadherin As an experimental method for indirectly confirming improvement of vascular permeability based thereon, a change in VE-cadherin was confirmed through a Western blot. Specifically, HUVECs were seeded into 6-well plates at a density of 3 ⁇ 10 5 per well, followed by incubation for 24 hours. Thereafter, single peptides and Fc-TPP were treated with 1 ⁇ M for 10 minutes to perform a Western blot. Gel subjected to SDS-PAGE was transported into PVDF membrane, a primary antibody (SantaCruz) and an HRP-conjugated secondary antibody (SantaCruz) which recognize VE-cadherin and ⁇ -actin, respectively were used for detection. ImageQuant LAS4000 mini (GE Healthcare) was used for analysis.
- FIG. 11 a illustrates a result of Western blot for identifying biological mechanism of Fc-TPP in HUVEC.
- FIG. 11 a in the case of Sema3A derived protein, when treating the control group VEGF165A and Sema3A, a decrease in VE-cadherin was observed. In the case of single peptides A22 and A22P, no change in VE-cadherin was observed. Also, in the case of Fc and Fc-4A22, VE-cadherin was not decreased, whereas in the case of Fc-15A22 and Fc-15A22P, VE-cadherin was decreased (left panel). In the case of Sema3F derived TPP, only the control group VEGF165A and Fc-15F22P induced a decrease in VE-cadherin (right panel).
- HUVECs were seeded into Transwell plate (Corning) at a density of 5 ⁇ 10 4 per well in a upper chamber using Endothelial Growth Medium, PromoCell (EGM) medium, followed by incubation at 37° C. for 3 days under 5% CO 2 condition. Thereafter, after changing the medium to Endothelial Basal Medium (EBM, PromoCell), the control group VEGF165A, Sema3A and Sema3F were treated with about 1.3 nM and single peptides and Fc-TPP with 1 ⁇ M for 30 minutes.
- EBM Endothelial Basal Medium
- Dextran-FITC (Sigma) 50 ⁇ g was put into the upper chamber and the medium of the lower chamber was sampled to measure its fluorescence after 30 minutes, using the principle that a fluorescent substance is observed in the lower chamber when the permeability in HUVECs was improved.
- FIG. 11 b illustrates a result of Transwell assay performed for confirming whether TPP improves permeability of HUVEC.
- FIG. 11 b it was confirmed that in the case of VEGF165A and Sema 3A, permeability of HUVEC was improved, and in the case of single peptides A22 and A22P, and Fc-4A22 with short Fc part and linker of TPP, the improvement of vascular permeability was not induced.
- Fc-15A22 and Fc-15A22P it was confirmed that permeability of HUVEC was improved.
- HUVEC permeability was not improved by Sema3F itself, but in the case of Fc-15F22P, the improvement of vascular permeability was induced. Also, in the case of single peptides F22 and F22P, and in the case of fusion forms with lower binding capacity, Fc-4F22 and Fc-15F22, permeability of HUVEC was not improved.
- A431 cells were injected into Balb/c nude mice with subcutaneous injection at a density of 5 ⁇ 10 6 per mouse, and when the tumor volume became about 300 to 400 mm 3 after about 9 days, each of PBS, Fc, Fc-15A22P and peptide A22P in an amount of 5 mg/kg was intravenously injected. 3 hours after injection, tumors were extracted from mice to conduct immunohistochemistry experiment. The extracted tumors were cut into 20 ⁇ m thick using a frozen section method, and stained with a primary antibody, CD31 antibody (BD Pharmingen), and a TRITC (red fluorescence)-conjugated secondary antibody. Further, in order to observe Fc-TPP distributed in the tissues, FITC (green fluorescence)-conjugated antibodies recognizing Fc were used.
- FIG. 11 c illustrates a result of immunohistochemistry for confirming TPP's infiltration capacity in actual tumor tissues.
- single peptides A22 and A22P did not infiltrate into the tissues, whereas, unlike the control group Fc, the fusion forms Fc-15A22 and Fc-15A22P selectively reached tumor tissues, and they infiltrated into tumor cells. Also, it was confirmed that Fc-15A22P having higher binding capacity to neuropilin-1 and -2 than Fc-15A22 infiltrated into tissues more effectively.
- Evans Blue assay was conducted.
- FaDu cells were injected into Balb/c nude mice (Nara Bio, 4-week-old, female) with subcutaneous injection at a density of 5 ⁇ 10 6 per mouse, and when the tumor volume became about 500 mm 3 after about 10 days, Evans Blue (Sigma) 1 mg, and each of PBS, Fc, and Fc-15A22P in an amount of 7.5 mg/kg was intravenously injected.
- VEGF165A 400 ng
- Semaphorin 3A 400 ng
- mice 40 minutes after injection, the mice were perfused through the heart with PBS containing 1% BSA, and tumor tissues were extracted ( FIG. 11 d ). Then, the extracted tissues were put into 1 ml of 2,2N-methylaformamide (Sigma) and reacted at 37° C. overnight under mild shaking condition. Thereafter, the supernatant was obtained from centrifugation to measure the absorbance (600 nm), and the amount that Evans Blue penetrated was quantified.
- 2,2N-methylaformamide Sigma
- FIG. 11 d illustrates a result of confirming the improvement of vascular permeability through Evans Blue assay.
- the left panel is a photograph of cancer cells extracted, and the right panel illustrates a result of measuring absorbance (600 nm) for quantifying the Evans Blue.
- absorbance 600 nm
- FIG. 11 e illustrates a result of Western blot for a change in E-cadherin in human head and neck cancer cell line FaDu.
- Sema3A derived TPP did not induce a change in E-cadherin
- Fc-15A22 and Fc-15A22P induced a decrease in E-cadherin.
- Sema3F induced a decrease in E-cadherin and when treated with Fc-15F22P, E-cadherin was decreased.
- Fc-TPP was constructed from peptides derived from each of Sema3A and Sema3F, but the original proteins and peptides derived therefrom did not always induce the same results in the properties of original proteins, specificity to neuropilin-1 or neuropilin-2, and tendency of signal transduction such as a decrease in VE-cadherin or E-cadherin. Further, peptides having binding capacity to neuropilin-1 and -2 did not induce signal transduction by TPP as single forms, whereas the fusion form Fc-TPP effectively induced signal transduction. This means that signal transduction by neuropilin-1 and -2 is effective in the fusion form of Fc-TPP.
- Fc-4A/F22 did not induce signal transduction
- Fc-15A/F22 and Fc-15A/F22P induced signal transduction. This means that the fusion form Fc-TPP more effectively induces signal transduction by neuropilin-1 and -2, than signal form peptides, and that the effect varies depending on the length of linkers of Fc-TPP.
- VEGF165A is known to form new blood vessels (angiogenesis) using neuropilin-1 as a co-receptor.
- angiogenesis in vitro based thereon, tube formation assay was conducted.
- 50 ⁇ l of ECMatrix was injected into 96-well plates and polymerized at 37° C. for 2 hours.
- HUVEC cells were suspended using Endothelial basal medium (EBM, PromoCell) and plated on ECMatrix at a density of 1 ⁇ 10 4 per well by mixing with VEGF165A (20 ng/ml), Fc, Fc-15A22P (1 ⁇ M), followed by incubation for 8 hours. The incubated cells were observed by a microscope to obtain images.
- EBM Endothelial basal medium
- FIG. 12 a illustrates a result confirming the capacity to inhibit tube formation through tube formation assay. As illustrated in FIG. 12 a , it was confirmed that VEGF165A increased tube formation as already known, and that Fc-15A22P, unlike Fc, inhibited the tube formation.
- matrigel plug assay was conducted.
- 7.5 ⁇ 10 6 A431 cells per mouse, 80 ⁇ g of Fc or Fc-15A22P, and 0.4 ml of Matrigel (BD Biosciences) were subcutaneously injected into 6 to 8-week old balb/c nude mice.
- matrigel plug was removed to take photographs of the image ( FIG. 12 b ), and cut into 20 ⁇ m thick using a frozen section method to perform immunohistochemistry experiments. Blood vessels were stained with a primary antibody, CD31 antibody, and a TRITC (red fluorescence)-conjugated secondary antibody recognizing the primary antibody, to measure the density of the blood vessels.
- FIG. 12 b illustrates a result confirming that Fc-15A22P is capable of inhibiting angiogenesis induced by VEGF165A in mice in vivo.
- Example 9 above confirmed that Fc-TPP improves the permeability of vascular endothelial cells in vitro and in vivo. Accordingly, TPP was fused to Fc terminus of antibodies as a format for verifying the effect of TPP in mouse models.
- FIG. 13(A) is a schematic diagram illustrating a fusion antibody (mAb-TPP) where a peptide binding to neuropilin is linked by a peptide linker to C-terminus of a heavy chain constant region of single clone antibody.
- FIG. 13(B) illustrates a schematic diagram illustrating that the fusion antibody of (A) binds to b1 domain of neuropilin membrane protein.
- FIG. 13(C) illustrates the mechanism expected when the fusion antibody (mAb-TPP) is introduced into the body.
- the peptide fused antibodies bind to overexpressed neuropilin membrane proteins of tumor endothelium and various tumor cells to ⁇ circle around (1) ⁇ increase selective distribution in tumor tissues, ⁇ circle around (2) ⁇ increase extravasation, and ⁇ circle around (3) ⁇ induce signal transduction which increases infiltration into tumor tissues, thereby remarkably increasing tumor tissue specific distribution of fusion antibodies and infiltration into tumor tissues.
- FIGS. 14 and 15 are maps of exemplary vectors for expressing IgG heavy chain-TPP and IgG light chain in host cells, respectively.
- FIGS. 16(A) and 17(A) are schematic diagrams illustrating TPP fused antibodies.
- the expression and purification were performed in HEK293F in the same manner as in Example 4 above, and the purity was confirmed through SDS-PAGE.
- FIGS. 16(B) and 17(B) illustrate results of analyzing the size and purity on SDS-PAGE under reducing and non-reducing conditions, after transient expression and purification in HEK293F cells through co-transformation.
- Table 8 shows the yield of proteins produced per 1 L culture of purified TPP fusion proteins.
- the obtained yields of proteins (Cetuximab-15A22p and Trastuzumab-15A22p) are not remarkably different from those of wild-type proteins (Cetuximab and Trastuzumab).
- CM5 chips In order to confirm through surface plasmon resonance (SPR) whether mAb-TPP maintains binding affinity with antigen binding capacity of the existing antibodies, EGFR and Her2 in an amount of about 1000 RU were fixed on CM5 chips.
- the analysis was performed with HBS-EP buffer (10 mM Hepes, 3 mM ethylenediamine tetra acetic acid, and 0.005% surfactant P20 (pH 7.4), GE Healthcare) flowing at a rate of 30 ⁇ l/min.
- the regeneration of CM5 chip was performed by streaming a buffer (20 mM NaOH, 1 M NaCl, pH 10.0) at a stream velocity 30 ⁇ l/min for 1 minute.
- Sandwich ELISA was performed to confirm bispecificity of mAb-TPP.
- mAb and mAb-TPP at a concentration of 100 nM were bound to b1b2 domains of neuropilin-1 and -2, and then washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA).
- bionylated EGFR SIGMA-ALDRICH co., USA
- Her2-ECD R&D systems, Minneapolis, Minn.
- PBST 0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA.
- FIGS. 16(C) and 17(C) are results confirming from ELISA analysis the bispecificity of mAb-TPP which has binding capacity to neuropilin-1 or -2 and original antigens of mAb simultaneously.
- PBST buffer pH 6.0, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, 0.005% surfactant P20, SIGMA-ALDRICH co., USA
- PBST buffer pH 6.0, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, 0.005% surfactant P20, SIGMA-ALDRICH co., USA
- streaming HBS-EP buffer (10 mM Hepes, 3 mM ethylenediamine tetra acetic acid, and 0.005% surfactant P20 (pH 8.0), GE Healthcare) at a stream velocity 30 ⁇ l/min for 1 minute.
- FIG. 18 illustrates a result confirming whether the binding capacities of mAb and mAb-TPP to FcRn are similar to each other depending on pH.
- mAb Cetuximab, Trastuzumab
- mAb-TPP Cetuximab-TPP, Trastuzumab-TPP
- FIG. 19 a illustrates a result of IHC for confirming the infiltration capacity of Cetuximab-TPP in tumor tissues.
- FIG. 19 a in the case of Cetuximab, green fluorescence was observed only around the blood vessels in the two cancer cell tissues of FaDu and A431.
- Cetuximab-15A22P infiltrated into the tissue to be further away from the blood vessels.
- the samples were compared after 3 hours and 12 hours, and accordingly it was confirmed that the cell infiltrates further into the tissue as time passes. ImageJ was used to quantify this.
- FIG. 19 b Western blot was performed from the cancer cell tissue used in this condition, and the result is illustrated in FIG. 19 b .
- FIG. 19 b when performing Western blot using antibodies recognizing the heavy chain site and light chain site of human antibody, respectively, it was confirmed that more amount of Cetuximab-15A22P was present in the tissue than Cetuximab. Also, it was confirmed that only E-cadherin was decreased by Cetuximab-TPP, and N-cadherin, vimentin, fibronetin, etc., which are proteins relating to metastasis of cancer cells, did not change.
- an IHC experiment was performed to confirm the antibody infiltrated into tumor tissue using the constructed Trastuzumab-15A22P.
- 5 ⁇ 10 6 cells of SK-OV-3 were subcutaneously injected into Balb/c nude mice, respectively, and when the volume of the tumor became about 300-400 mm 3 after about 9 days, 2.5 mg/kg of PBS, Trastuzumab and Trastuzumab-15A22P were intravenously injected, respectively.
- the tumor was extracted from the mice after 3 hours and 12 hours, respectively, and an immunohistochemistry experiment was performed. The tissue was stained and observed in the same manner as the experiment above.
- FIG. 20 a illustrates a result of IHC for confirming the infiltration capacity of Trastuzumab-TPP in tumor tissues.
- FIG. 20 a in the case of Trastuzumab, green fluorescence was observed only around the blood vessels.
- Trastuzumab-15A22P in the case of Trastuzumab-15A22P, as compared to Trastuzumab, it is confirmed that Trastuzumab-15A22P infiltrated into the tissue to be further away from the blood vessels.
- the samples were compared after 3 hours and 12 hours, and accordingly it was confirmed that the cell infiltrates further into the tissue as time passes. ImageJ was used to quantify this.
- FIG. 20 b Western blot was performed from the cancer cell tissue used in this condition, and the result is illustrated in FIG. 20 b .
- FIG. 20 b when performing Western blot using antibodies recognizing the heavy chain site and light chain site of human antibody, respectively, it was confirmed that more amount of Trastuzumab-15A22P was present in the tissue than Trastuzumab. Also, it was confirmed that only E-cadherin was decreased by Trastuzumab-TPP, and N-cadherin, vimentin, fibronetin, etc., which are proteins relating to metastasis of cancer cells, did not change. Through this, it was indirectly proved that metastasis and infiltration of cancer cells do not occur by the effect of TPP.
- FIG. 20 c illustrates a result of measuring the volume of the tumor to confirm the effect of Trastuzumab-TPP to inhibit cancer cell growth. As illustrated in FIG.
- FIG. 20 d illustrates a result of measuring the weight of mice during the experiment. As illustrated in FIG. 20 d , it can be understood that the experimental group Trastuzumab-15A22P did not present a great difference in weight as compared with the experimental group Trastuzumab, and it is determined to have no toxicity.
- tumor tissue-penetrating A22p peptide may be generally applied to various monoclonal antibodies recognizing various antigens.
- 1 ⁇ 10 4 cells (FaDu, SK-OV-3) were diluted in 0.5 ml of a medium including 10% FBS per well in 24-well plate, respectively, and cultured.
- the cells were stabilized, they were treated with 1 ⁇ M of Fc, Fc-15A22P, mAb, and mAb-15A22P, and observed for 72 hours, and then the number of cells alive was counted to compare the degree of cell growth.
- the effect was confirmed by treating cell line FaDu with Fc, Fc-15A22P, Cetuximab, and Cetuximab-15A22P, and treating SK-OV-3 with Fc, Fc-15A22P, Trastuzumab, and Trastuzumab-15A22P.
- FIG. 21 illustrates a result of in vitro evaluation on the degree of cell growth inhibition by treating FaDu and SK-OV-3 with Fc-TPP and mAb-TPP.
- Cetuximab and Cetuximab-15A22P inhibited cell growth by about 30% in cell line FaDu
- Trastuzumab and Trastuzumab-15A22P inhibited cell growth by about 30-35% in cell line SK-OV-3. It was confirmed that there was no difference between the mAb and mAb-TPP.
- Fc and Fc-TPP did not inhibit cell growth in both FaDu and SK-OV-3.
- TPP maximizes the effect by increasing infiltration into tumor tissue without directly affecting the growth of cancer cells. Also, it was indirectly confirmed that the effect of mAb-TPP confirmed through the above test is not an effect by the interaction between EGFR and neuropilin, and between HER2 and neuropilin.
- An aspect of the present invention provides a tumor tissue-penetrating peptide (TPP) specifically binding to neuropilin.
- TPP tumor tissue-penetrating peptide
- the tumor tissue-penetrating property in the above aspect means, for example, having any one of the properties of 1) specifically recognizing a tumor specific vascular endothelial cell, a tumor cell or tissue and accumulating in it, 2) widening intercellular gaps between tumor vascular endothelial cells and promoting extravasation, and 3) controlling intercellular gap between tumors within the tumor and increasing infiltration within the tumor.
- neuropilin as used herein is a transmembrane glycoprotein, and there are two forms of neuropilin, NRP1 and NRP2.
- the structures of NRP1 and NRP2 are as illustrated in FIGS. 1 (D) and (E).
- Neuropilin broadly consists of five domains, and from the N-terminus, a1 and a2 domains are classified as CUB domains, and an Ig-like C2 type of semaphorin binds thereto. Particularly, this site forms a complex with plexin, and plays a role of increasing the binding force with semaphorin-plexin.
- the b1 and b2 domains of neuropilin are classified as FV/VIII domains, and the C-terminus of VEGF family ligand or secreted class 3 semaphorin ligands (secreted Sema3s) binds thereto.
- the VEGF ligand and Sema3s have a site recognizing furin hydrolysis enzymes (RXRR, Arg-X-Arg-Arg), and thus they commonly end with an arginine (Arg) amino acid residue at the C-terminus by Furin processing (Adams et al. 1997).
- the tumor tissue-penetrating peptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4 is a peptide designed based on the similarity of the C-terminus sequences by analyzing the sequence of the VEGF165A and the furin C-terminus sequence of semaphorin 3A and semaphorin 3F known to bind to neuropilin, by analyzing the amino acid sequence and length of the binding site of VEGF165A ligand binding to b1b2 domains of neuropilin.
- A22 which is a tumor tissue-penetrating peptide represented by an amino acid sequence of SEQ ID NO:1 is a peptide consisting of 22 amino acids derived from residues 739-760, which are part of the basic domain of semaphorin 3A.
- A22p which is a tumor tissue-penetrating peptide represented by an amino acid sequence of SEQ ID NO: 2, transforms the third amino acid from the C-terminus of A22, which is the 758th asparagine (Asn758), to proline (Pro, P) (Asn758Pro).
- F22 which is a tumor tissue-penetrating peptide represented by an amino acid sequence of SEQ ID NO: 3, is a peptide consisting of 22 amino acids derived from residues 758-779, which are part of the basic domain of semaphorin 3F.
- F22p which is a tumor tissue-penetrating peptide represented by an amino acid sequence of SEQ ID NO: 4, transforms the third amino acid from the C-terminus of F22, which is the 777th asparagine (Asn777), to proline (Pro, P) (Asn777Pro).
- the peptides A22p and F22p are designed to derive peptides with improved affinity with neuropilin by inserting mutants to Sema3A- and Sema3F-derived peptides.
- the tumor tissue-penetrating peptide of the above aspect may further include a linker peptide.
- the linker peptide may consist of 1 to 50 amino acids, preferably 4 to 20 amino acids, and more preferably 4 to 15 amino acids.
- the linker peptide may consist of glycine, serine or alanine, preferably the sequence of the linker peptide may consist of an amino acid sequence of (GA)n or (GGGGS)m (wherein n and m are each independently an integer between 1 and 20), and more preferably may consist of an amino acid sequence of GAGA or (GGGGS)3.
- the tumor targeting capacity is confirmed in various combinations of the linker and semaphorin-derived sequence in a detailed embodiment of the present invention.
- the name of peptide, sequence of linker, semaphorin-derived sequence, length of entire amino acid and SEQ ID NO. are as shown below.
- Another aspect of the present invention provides a fusion protein, a nanoparticle or a liposome having the tumor tissue-penetrating peptide fused therein.
- the protein may be antibodies, antibody fragments, immuoglubulin, peptides, enzymes, transcription factors, toxins, antigen peptides, hormones, carrier proteins, structural proteins, motor function proteins, receptors, signaling proteins, storage proteins, membrane proteins, transmembrane proteins, internal proteins, external proteins, secretory proteins, viral proteins, native proteins, glycoproteins, cleaved proteins, proteins with disulfide bond, protein complexes, chemically modified proteins, or prions, etc.
- liposomes include at least one lipid bilayer enclosing the inner aqueous compartment, which is capable of being associated by itself.
- Liposomes may be characterized by membrane type and size thereof.
- Small unilamellar vesicles SUVs
- Large unilamellar vesicles LUVs
- Oliglamellar large vesicles and multilamellar large vesicles may have multiple, usually concentric, membrane layers and may be at least 100 nm in diameter.
- Liposomes with several nonconcentric membranes, i.e., several small vesicles contained within a larger vesicle are referred to as multivesicular vesicles.
- a nanoparticle refers to a particle including substances ranging between 1 and 1,000 nm in diameter.
- the nanoparticle may be a metal nanoparticle, a metal/metal core shell complex consisting of a metal nanoparticle core and a metal shell enclosing the core, a metal/non-metal core shell consisting of a metal nanoparticle core and a non-metal shell enclosing the core, or a non-metal/metal core shell complex consisting of a non-metal nanoparticle core and a metal shell enclosing the core.
- the metal may be selected from gold, silver, copper, aluminium, nickel, palladium, platinum, magnetic iron and oxides thereof, but is not limited thereto, and the non-metal may be selected from silica, polystyrene, latex and acrylate type substances, but is not limited thereto.
- the tumor tissue-penetrating peptide may have at least a bivalent binding to neuropilin.
- fusion refers to unifying two molecules having the same or different function or structure
- the methods of fusing include any physical, chemical or biological method binding the tumor tissue-penetrating peptide to the protein, nanoparticle or liposome.
- the fusion may be made by a linker peptide, and for example, the linker peptide may bind to C-terminus of Fc fragment of an antibody.
- a complete antibody has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to each heavy chain by a disulfide bond (SS-bond).
- a constant region of the antibody is divided into a heavy-chain constant region and a light-chain constant region, and the heavy-chain constant region has ⁇ , ⁇ , ⁇ , ⁇ and ⁇ types, and ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 1 and ⁇ 2 subclasses.
- the light-chain constant region has ⁇ and ⁇ types.
- variable chain as used herein may be interpreted to include a full-length heavy chain including variable region domain VH including an amino acid sequence having a variable region sequence sufficient to confer antigen-specificity and three constant region domains CH1, CH2 and CH3, and a fragment thereof.
- light chain as used herein may be interpreted to include a full-length light chain including a variable region domain VL including an amino acid sequence having a variable region sequence sufficient to confer antigen-specificity and constant region domain CL, and a fragment thereof.
- a fragment of an antibody refers to each domain of a heavy chain or a light chain of an antibody, or a fragment thereof.
- it may be a heavy-chain constant region, a heavy-chain variable region, a light-chain constant region, or a light-chain variable region of an antibody, or a fragment thereof.
- the fragment of the antibody may be a heavy-chain constant region of an antibody.
- the fragment of the antibody may be a monomer, a dimer or a polymer.
- the antibody includes monoclonal antibodies, non-specific antibodies, non-human antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFV) and anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of these antibodies, but is not limited thereto.
- the monoclonal antibody may be IgG, IgM, IgA, IgD or IgE.
- the monoclonal antibody may be IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type, and may be IgG1.
- the light-chain constant region of the antibody may be ⁇ or ⁇ type.
- the peptide may bind to a heavy-chain constant region (Fc) fragment of an antibody, preferably to the C-terminus of a heavy-chain constant region (Fc) fragment of an antibody, and the binding may be formed by a linker peptide.
- Fc heavy-chain constant region
- Another aspect of the present invention provides a polynucleotide coding a peptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 10.
- polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer present in single-stranded or double-stranded form. It includes RNA genome sequence, DNA (gDNA and cDNA), and RNA sequence transcribed therefrom. Unless otherwise described, it also includes an analog of the natural polynucleotide.
- the polynucleotide includes not only a nucleotide sequence coding an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 10, but also a complementary sequence thereto.
- the complementary sequence includes a sequence fully complementary and a sequence substantially complementary. For example, this means a sequence that may be hybridized with a nucleotide sequence coding an amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 10 under stringent conditions known in the pertinent art.
- the polynucleotide may be modified.
- the modification includes the addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.
- the polynucleotide coding the amino acid sequence is interpreted to include a nucleotide sequence that has substantial identity to the nucleotide sequence.
- the substantial identity may refer to a sequence having at least 80% sequence identity, at least 90% sequence identity, or at least 95% sequence identity when aligning the nucleotide sequence to correspond to any other sequence as much as possible and analyzing the aligned sequence using an algorithm generally used in the pertinent art.
- Another aspect of the present invention provides a recombinant vector including the polynucleotide.
- vector refers to a means for expressing a target gene in a host cell.
- the vector may include plasmid vector, cosmid vector, bacteriophage vector, and virus vectors such as adenovirus vector, retrovirus vector, and adeno-associated virus vector.
- the vector that may be used as the recombinant vector may be produced by operating plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), phages (for example, ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1 and M13, etc.), or virus (for example, CMV, SV40, etc.) commonly used in the pertinent art.
- plasmid for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGE
- a polynucleotide coding an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 10 in the recombinant vector may be operatively linked to a promoter.
- operatively linked means a functional linkage between a nucleotide expression control sequence (such as a promoter sequence) and a second nucleotide sequence. Accordingly, the control sequence may control the transcription and/or translation of the second nucleotide sequence.
- the recombinant vector may be generally constructed as a vector for cloning or a vector for expression.
- vector for expression vectors generally used for expressing foreign protein from plants, animals or microorganisms in the pertinent art may be used.
- the recombinant vector may be constructed by various methods known in the pertinent art.
- the recombinant vector may be constructed to be a vector that employs a prokaryotic cell or an eukaryotic cell as a host.
- the vector when the vector used is an expression vector and employs a prokaryotic cell as a host, the vector generally includes a strong promoter which may promote transcription (for example, pL ⁇ promoter, tip promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and termination sequences for transcription/translation.
- a strong promoter which may promote transcription (for example, pL ⁇ promoter, tip promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and termination sequences for transcription/translation.
- a replication origin operating in the eukaryotic cell included in the vector may include an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, a CMV replication origin and a BBV replication origin, etc., but is not limited thereto.
- a promoter derived from a genome of a mammal cell for example, a metalthionine promoter
- a promoter derived from a virus of a mammal cell for example, an adenovirus anaphase promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a cytomegalo virus (CMV) promoter, or a tk promoter of HSV
- the promoter generally has a polyadenylated sequence as a transcription termination sequence.
- the vector may express an antibody having the peptide fused therein or a fragment thereof.
- the vector may use both a vector system expressing a peptide and an antibody or fragment thereof simultaneously in one vector, or a vector system expressing them in separate vectors. For the latter, the two vectors may be introduced into the host cell through co-transformation and targeted transformation.
- the recombinant vector of the present invention may have the cleavage map illustrated in FIG. 4(B) or FIG. 13(B) .
- Another aspect of the present invention provides a host cell transformed with the recombinant vector.
- a prokaryotic cell examples include strains belonging to the genus Bascillus such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W 3110 , Bascillus subtilus and Bascillus thuringiensis, Salmonella typhimurium , intestinal flora and strains such as Serratia marcescens and various Pseudomonas Spp., etc.
- Bascillus such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W 3110 , Bascillus subtilus and Bascillus thuringiensis
- Salmonella typhimurium , intestinal flora and strains such as Serratia marcescens and various Pseudomonas Spp
- a host cell such as Saccharomyce cerevisiae , an insect cell, a plant cell, and an animal cell, for example, SP2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RN, and MDCK cell line, etc.
- SP2/0 CHO (Chinese hamster ovary) K1
- CHO DG44 CHO DG44
- PER.C6, W138 BHK, COS-7, 293, HepG2, Huh7, 3T3, RN
- MDCK cell line etc.
- Another aspect of the present invention provides a method for preparing a tumor tissue-penetrating peptide, including culturing the host cell.
- the polynucleotide or a recombinant vector including the same may be inserted into a host cell using an insertion method well known in the pertinent art.
- the transfer may be carried out according to CaCl 2 method or an electroporation method, etc.
- the vector may be transferred into a host cell according to a microscope injection method, calcium phosphate precipitation method, an electroporation method, a liposome-mediated transformation method, and a gene bombardment method, etc., but the transferring method is not limited thereto.
- the product ability is higher than using animal cells.
- it is not suitable for production of intact Ig form of antibodies due to glycosylation, it may be used for production of antigen binding fragments such as Fab and Fv.
- the method for selecting the transformed host cell may be readily carried out according to a method well known in the pertinent art using a phenotype expressed by a selected label.
- the selected label is a specific antibiotic resistance gene
- the transformant may be readily selected by culturing the transformant in a medium containing the antibiotic.
- the aspect is a concept including the preparation of a tumor tissue-penetrating peptide (TPP), and an antibody having the peptide fused therein or a fragment thereof.
- TPP tumor tissue-penetrating peptide
- an aspect of the present invention provides a composition for treating or preventing cancer or angiogenesis-related diseases, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- the tumor tissue-penetrating peptide of the present invention specifically binds to neuropilin, thereby being specifically distributed in tumors, and exhibiting efficacy to penetrate into tumors. Also, a specific part is substituted with a sequence of vascular endothelial growth factor A (VEGF165A), thereby maintaining tumor infiltration effect and remarkably improving affinity to neuropilin. Also, the tumor tissue-penetrating peptide of the present invention allows the removal of heparin binding site, thereby minimizing nonspecific binding.
- VEGF165A vascular endothelial growth factor A
- the antibody having the tumor tissue-penetrating peptide fused therein of the present invention shows a production yield similar to wild-type antibody, and has the property of a bispecific antibody that can target two types of antigens, an antigen to which the antibody binds and neuropilin to which the tumor tissue-penetrating peptide binds. Accordingly, it may allow an antibody to reach a tumor tissue with high efficiency, and thus is expected to have a high effect in treating cancer.
- an antibody having the tumor tissue-penetrating peptide fused therein or a fragment thereof maintains the antigen binding capacity which the wild-type antibody originally has, unique function of heavy-chain constant region (Fc), i.e., binding with Fc ⁇ Rn (neonatal Fc receptor) and FcRs (Fc gamma receptors), and accordingly has a long serum half-life. Also, it has an advantage that the binding site (protein A and protein G) during the purification process is preserved, and the antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity may be maintained.
- Fc heavy-chain constant region
- the tumor tissue-penetrating peptide specifically binds to neuropilin, and competes with VEGF165A binding to neuropilin. Accordingly, it inhibits the angiogenesis function caused by VEGF165A binding to neuropilin, and thus is expected to have an effect of treating both cancer and angiogenesis-related diseases.
- the cancer may be selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of lung, squamous cell carcinoma of lung, peritoneal cancer, skin cancer, skin or ocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, pancreatic cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, liver cancer and head and neck cancer.
- the angiogenesis-related disease may be selected from the group consisting of diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginalkeratolysis, trauma, rheumatoid arthritis, systemic lupus, polyarteritis, Wegeners sarcoidosis,
- the composition may include a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier included in the composition may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, minute crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil, etc., but are not limited thereto.
- the pharmaceutical composition may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspension, a preservative, etc.
- the pharmaceutical composition for preventing or treating cancer or angiogenesis-related diseases may be administered orally or parenterally.
- a parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, nasal administration, intrapulmonary administration, intrarectal administration, etc.
- a composition for oral administration is formulated to coat an active substance or to be protected against degradation in stomach.
- the pharmaceutical composition may be administered by any device which can transport active substances to target cells.
- Proper dose of the pharmaceutical composition for preventing or treating cancer or angiogenesis-related diseases may vary according to various factors such as method for formulating, administration method, age, weight, gender, pathological state of patient, food, administration time, administration route, excretion rate and reaction sensitivity, etc.
- a proper dose of the composition is within the range of 0.001 and 100 mg/kg based on an adult.
- pharmaceutically effective dose refers to an amount sufficient to prevent or treat cancer or angiogenesis-related diseases.
- the composition may be formulated with pharmaceutically acceptable carriers and/or excipients according to a method that can be easily carried out by those skilled in the art, and may be provided in a unit-dose form or enclosed in a multiple-dose vial.
- the formulation may be in the form of a solution, a suspension, syrup or an emulsion in oily or aqueous medium, or may be extracts, powders, granules, tablets or capsules, and may further include a dispersion agent or a stabilizer.
- the composition may be administered individually or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
- the composition includes an antibody or an antigen-binding fragment, and thus may be formulated into immuno liposome.
- Liposome including an antibody may be prepared according to a method well known in the pertinent art.
- the immuno liposome is a lipid composition including phosphatidylcholine, cholesterol and polyethyleneglycol-derived phosphatidylethanolamine, and may be prepared by reverse phase evaporation method.
- a Fab′ fragment of antibody may be conjugated to liposome through disulphide exchange reaction.
- Liposome may further include chemical therapeutic agents such as Doxorubicin.
- an aspect of the present invention provides a composition for diagnosing cancer, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- Diagnosing refers to demonstrating the presence or characteristic of a pathophysiological condition. Diagnosing in the present invention refers to demonstrating the onset and progress of cancer.
- the tumor tissue-penetrating peptide may bind to a fluorescent substance for molecular imaging in order to diagnose cancer through images.
- the fluorescent substance for molecular imaging refers to all substances generating fluorescence.
- red or near-infrared fluorescence is emitted, and more preferably, a fluorescence with high quantum yield is emitted.
- the fluorescence is not limited thereto.
- the fluorescent substance for molecular imaging is a fluorescent substance, a fluorescent protein or other substances for imaging, which may bind to the tumor tissue-penetrating peptide, but is not limited thereto.
- the fluorescent substance is fluorescein, BODYPY, tetramethylrhodamine, Alexa, cyanine, allopicocyanine, or a derivative thereof, but is not limited thereto.
- the fluorescent protein is Dronpa protein, enhanced green fluorescence protein (EGFP), red fluorescent protein (DsRFP), Cy5.5, which is a cyanine fluorescent substance presenting near-infrared fluorescence, or other fluorescent proteins, but is not limited thereto.
- EGFP enhanced green fluorescence protein
- DsRFP red fluorescent protein
- Cy5.5 Cy5.5
- other substances for imaging are ferric oxide, radioactive isotope, etc., but are not limited thereto, and they may be applied to imaging equipment such as MR, PET.
Abstract
Description
- The present invention relates to a tumor tissue-penetrating peptide (TPP) specifically binding to neuropilin.
- Also, the present invention relates to a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the tumor tissue-penetrating peptide fused therein.
- Also, the present invention relates to a polynucleotide coding the tumor tissue-penetrating peptide, a recombinant vector including the same, a host cell transformed with this vector, and a method for preparing a tumor tissue-penetrating peptide using the host cell.
- Also, the present invention relates to a pharmaceutical composition for treating or preventing cancer, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- Also, the present invention relates to a composition for diagnosing cancer, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- In the development research of antibodies for treatment, a hybridoma technology capable of producing monoclonal antibodies was developed about 35 years ago. Additionally, chimeric/humanized antibodies overcoming immunogenicity of mouse antibodies (HAMA; Human anti-mouse antibody response) initially received its clinical approval in 1997. In addition, complete human antibody, Humira received approval in 2003. Furthermore, in order to increase treatment efficacy, the research on bispecific antibodies, antibody drug conjugation (ADC), and long-lasting antibodies improving heavy-chain constant region (Fc) has been actively conducted.
- In the case of antibodies for treating a solid tumor, during a process where the antibodies are transferred to tumor tissues, the amount of antibodies transferred to the tumor tissues in actual bodies of human beings is merely 0.01 to 0.001% of the amount injected by various barriers, which means that the treatment effect of antibodies is very limited (Thurber et al. 2008). Accordingly, the development of antibody technology allowing the antibodies to be selectively accumulated in the tumor tissues and to have high permeability into the tumor tissues may increase the treatment effect of antibodies and thus is very important.
- There are two major reasons why the antibodies are not well permeated into tissues: 1) intrinsic properties of antibodies (size, antigen-binding properties, etc.) (Thurber and Dane Wittrup, 2012) and 2) fine physiological properties of tumor tissues which are different from normal tissues (Jain and Stylianopoulos, 2010).
- Since antibodies are big molecules of 150 kDa consisting of 12 domains, the antibodies in the blood are difficult to be transferred to the tumor tissues through diffusion or convection (Baker et al. 2008). Thus, among the research conducted to solve this, there was an attempt to administer only the domain which binds to an antigen of antibody. In the case of single-chain antibody fragment (scFv, 30 kDa) and heavy-chain variable region (VHH, 14 kDa), they permeated into the tumor tissues much more than the antibodies themselves. However, as their size decrease, a great deal thereof come out through the kidney, and thereby a half-life gets shorter. Thus, efficacy of antibodies has not been significantly improved (Behr et al. 1998).
- Another reason why the antibodies are not distributed in a large amount in the tissues is an antigen binding capacity of antibodies. The antibodies for treating the solid tumor are over-expressed in a tumor-associated antigen, or in a tumor, and have a high affinity to a target which is important for the tumor's growth. Even when the antibodies reach the tissues where a specific antigen is present, in the tumor tissues consisting of cells with a great amount of antigen expression, the antibodies are stayed in the antigen while binding to the antigen, due to their high affinity (Lee and Tannock, 2010). Also, after the binding, the antibodies penetrated into the cells along with antigens (Endocytosis) and are lysed. Consequently, the antibodies cannot exert their anti-cancer effects. In order to overcome this, the research for adjusting affinity or lengthening half-life has been processed (Dennis et al. 2007).
- Physiological properties of tumor tissues which prevent the antibodies from being permeated and distributed in the tumor tissues may be broadly classified into 4 cases, which are endothelial barrier, high interstitial fluid pressure, stromal impediment, and epithelial barrier.
- For the endothelial barrier, the tumor over-expresses and secretes factors (pro-angiogenic factor) which promote the growth of vascular endothelial cells located around blood vessel in order to receive a great deal of nutrients according to the tumor's rapid speed of growth. Accordingly, a large amount of new blood vessels are produced in an uneven manner, which leads to a decrease in the speed of entire blood flow. In order to overcome this, there is a method for increasing extravasation so that therapeutic agents could come out of the blood vessel and be distributed into the tissue. There is a case of enhancing a drug delivery into tumor tissue by co-administering TNF-α and IL02, which are cytokine inflammatory responses related with extravasation, chemical substance promoting extravasation (promoter chemical drug) (Marcucci et al. 2013), iRGD peptide, etc. in combination with therapeutic agents. However, these attempts were difficult to be commercialized and clinically experimented in that it is required to produce two substances of antibodies and extravasation promoter. Additionally, iRGD peptide reached its limit in that it needs to be administered in a great quantity (2 mg/kg or 4 mg/kg) (Sugahara et al. 2010).
- High tumor interstitial fluid pressure results from a situation where the pressure difference allowing the drug to be convected from the blood vessel to the tissue is small, or where the fluid pressure of tissue is higher than that of blood. High tumor interstitial fluid pressure is mainly caused due to the accumulation of interstitial fluid pressure in the absence of lymphatic duct in the tumor tissue, unlike in the normal tissue, and contributes to abnormal angiogenesis. In order to overcome this, a method for preventing the operation of a factor promoting the growth of vascular endothelial cell, particularly vascular endothelial cell growth factor-A (VEGF-A), and inhibiting angiogenesis to normalize the blood vessel, or a method for increasing the fluid pressure of blood vessel has been attempted. With regard to the method for increasing the fluid pressure of blood vessel, there was a case where plasma protein albumin was administered in combination with antibodies to increase osmotic pressure of blood vessel, thereby improving delivery effect of antibodies to the tumor tissue (Hofmann et al. 2009).
- The stromal impediment, which is an extracellular matrix barrier met when the antibodies come out to micro-vessels and are convected to the tissue, mainly consists of collagen and hyaluronan. The extracellular matrix greatly affects the shape of tumor. Accordingly, there is a big difference between the area where the drug is well distributed and the area where the drug is not well distributed, so drug distribution becomes uneven. Additionally, as an amount of expression of extracellular matrix increases, the tumor interstitial fluid pressure due to a high cell density with solid tumor stress (solid stress) increases. As the method for overcoming this, there is a method for inducing apoptosis of tumor tissue cell to reduce tumor interstitial cell density. Additionally, there was a case increasing the drug delivery effect about two times compared to a control group by processing an enzyme (collagenase) dissolving collagen in the tumor tissue to reduce solid stress (Eikenes et al. 2004).
- In the epithelial barrier, intercellular adhesion factors of tumor interstitial epithelial cell densely fill up an intercellular space, and thus they prevent the therapeutic agent from being diffused and convected between the cells. E-cadherin is well known as a main factor of the intercellular adhesion. Since a substance reducing the E-cadherin was found in virus (adenovirus-3), there was a case where only a part (JO-1) with an activity of reducing E-cadherin of cell among proteins constituting the virus was administered in combination with the antibody, thereby increasing an anti-cancer effect of antibody (Beyer et al. 2011).
- Considering the methods suggested so far to readily deliver the therapeutic agents to the tumor, most cases simply administer the therapeutic agents in combination with the substance for delivering this therapeutic agents well to the tumor tissue. Particularly, peptide is pharmacokinetics resulting from a small size of molecule, and has very short half-life. Thus, a great amount of peptide needs to be administered to actual patients and the administration needs to be frequently made. Furthermore, since the therapeutic agents and substances for tumor permeation operation need to be produced, respectively, which is an inevitable process during co-administration, its industrial practicability is low. Also, the peptide sequence and protein which do not exist in the natural world are likely to cause immunogenicity. Thus, ideally, it is required to develop a format where the antibody acquires tissue permeability as it is so that the delivery effect of one antibody molecule into the tumor tissue could be increased.
- Among the proteins existing in the natural field, the vascular endothelial cell growth factor-A (VEGF-A) is well known for inducing blood spout (extravasation), which is also called as a vascular permeability factor. This action is known as a phenomenon caused by the binding with a vascular endothelial cell growth factor receptor (VEGFR2). Interestingly, in a mutant experiment of the vascular endothelial cell growth factor-A, even if the factor is not combined to the vascular endothelial cell growth factor receptor, vascular permeability increased, which suggests that another receptor of the vascular endothelial cell growth factor-A exists (Stacker et al. 1999). Other professionals of the same age found that this receptor is neuropilin (NRP).
- Neuropilin was first found in a Xenopus nervous system. Neuropilin is a transmembrane glycoprotein, and has two types of NRP1 and NRP2. Neuropilin is expressed very weakly in normal cells, whereas is over-expressed in tumor vascular endothelial cells, solid tumor cells, blood tumor cells. Neuropilin operates as a coreceptor of VEGFRs (VEGF receptors) by binding to VEGF family ligands. Especially, NRP1 operates as a coreceptor of VEGFR1, VEGFR2 and VEGFR3, and binds to various VEGF ligands, thereby contributing to angiogenesis, cell survival, migration & adhesion, invasion, etc. in the tumor tissue. In comparison, NRP2 operates as a coreceptor of VEGFR2 and VEGFR3, thereby contributing to lymphangiogenesis and cell adhesion. Additionally, NRP1/NRP2 (NRP1/2) operates as a coreceptor of plexin family receptors to bind to secreted
class 3 semaphorins (Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, Sema3F and Sema3G). Since the neuropilin has no domain in functional cells, even if a ligand is binding thereto, the neuropilin has no activity by itself. It is known that signals are transferred through the VEGF receptor, which is the coreceptor, or through the plexin co-receptor. Sema3 binds to neuropilin and the plexin receptor at a ratio of 2:2:2 and operates. - There are cases reported that the operation of neuropilin and coreceptor is inhibited even when the neuropilin alone is targeted. For example, in the case of anti-neuropilin-1 antibody, it is reported that the anti-neuropilin-1 antibody is competitive to bind only to neuropilin-1 with VEGA-A, which is well known for binding to VEGFR2 and neuropilin-1, and has the function of inhibiting angiogenesis, cell survival, migration & adhesion and invasion, which are operations of VEGFR2 (Pan Q et al 2007). In the case of anti-neuropilin-2 antibody, it is reported that anti-neuropilin-2 antibody is competitive to bind to neuropilin-2 with VEGA-C which is well known for binding to VEGFR3 and neuropilin-2 at the same time, and has the function of inhibiting lymphangiogenesis and cell adhesion, which are operations of VEGFR3 (Cant M et al. 2008).
- Thus, the present inventors infer a part of minimum Sema3A- or Sema3F-derived peptide with an enhancing effect of vascular endothelial cell permeability by the interaction between the neuropilin and Sema3A or Sema3F, and designed a mutant peptide so as to have a high affinity with the neuropilin. The present inventors designed bivalent shape by converging peptide into a heavy-chain C-terminus of antibody so that the peptide could copy the function of Sema3A/Sema3F operating as a homodimer. As this design is a single molecule fused with antibody-peptide, a unique function of antibody is maintained as it is. Additionally, by promoting the antibody's accumulation into the tumor tissue and permeability into the tumor tissue by the binding to the neuropilin, and additionally inhibiting the operation of neuropilin coreceptor by the neuropilin targeting, a fusion antibody technology with an anti-angiogenesis effect has been developed.
- It is an aspect of the present invention to provide a tumor tissue-penetrating peptide (TPP) specifically binding to neuropilin.
- It is another aspect of the present invention to provide a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the tumor tissue-penetrating peptide fused therein.
- It is another aspect of the present invention to provide a polynucleotide coding the tumor tissue-penetrating peptide, a recombinant vector including the same, a host cell transformed with this vector, and a method for preparing a tumor tissue-penetrating peptide using the host cell.
- Also, it is another aspect of the present invention to provide a pharmaceutical composition for treating or preventing cancer or angiogenesis-related diseases, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- Also, it is another aspect of the present invention to provide a composition for diagnosing cancer or angiogenesis-related diseases, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- An aspect of the present invention provides a tumor tissue-penetrating peptide (TPP) specifically binding to neuropilin.
- Another aspect of the present invention provides a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the tumor tissue-penetrating peptide fused therein.
- Another aspect of the present invention provides a polynucleotide coding the tumor tissue-penetrating peptide, a recombinant vector including the same, a host cell transformed with this vector, and a method for preparing a tumor tissue-penetrating peptide using the host cell.
- Also, another aspect of the present invention provides a pharmaceutical composition for treating or preventing cancer or angiogenesis-related diseases, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- Also, another aspect of the present invention provides a composition for diagnosing cancer or angiogenesis-related diseases, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- The tumor tissue-penetrating peptide and a protein having the peptide fused therein of the present invention have the properties of specifically binding to neuropilin, and accordingly specifically accumulate in tumor tissues, widen intercellular gap between the tumor vascular endothelial cells to promote extravasation, and control intercellular gap between tumors within the tumor tissue to increase infiltration within tumor tissue to show a remarkably increased in vivo tumor-suppressing activity. Further, when a fusion antibody having Sema3A- and Sema3F-derived peptides and peptides with remarkably improved affinity of their wild-type peptides with neuropilin fused therein or in the antibody fragment are administered in the same dosage as a control group antibody having no peptide fused therein, they specifically accumulate in tumor tissues, and increase infiltration within tumor tissue, to show a remarkably increased in vivo tumor-suppressing activity, compared with the control group antibody.
- An antibody having the tumor tissue-penetrating peptide of the present invention fused therein or a fragment thereof has the property of a bispecific antibody which has a binding capacity with neuropilin to which a tumor tissue-penetrating peptide binds, while maintaining the antigen binding capacity which the antibody originally has, and accordingly accumulates a fusion antibody with high efficiency in tumor tissues and increases infiltration within tumor tissues and thus is expected to have a high effect in treating and diagnosing tumor. Also, a fragment of the antibody having the tumor tissue-penetrating peptide of the present invention fused therein has the property of inhibiting angiogenesis by VEFG165A by inhibiting VEFG165A from binding to
neuropilin 1/2, and thus is expected to be used for treating and diagnosing various diseases such as diabetic retinopathy, rheumatoid arthritis, or atherosclerosis, relating to angiogenesis. - An antibody having the peptide of the present invention fused therein or a fragment thereof shows a production yield similar to wild-type antibody which does not have a peptide fused therein, and thus there is no problem in mass production. Also, the fusion antibody or a fragment thereof maintains the antigen binding capacity which the wild-type antibody originally has, unique function of heavy-chain constant region (Fc), i.e., binding to FcRn (neonatal Fc receptor), and accordingly has a long serum half-life. Also, it has an advantage that the binding site (protein A and protein G) is preserved during the purification process, and the antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity may be maintained.
-
FIG. 1 is a schematic diagram illustrating the structures of semaphorins, VEGF165A, and neuropilin-1 and -2. - In
FIGS. 1(A) and 1(B) , semaphorin broadly includes three domains. From the N-terminus, a sema domain is a site binding to plexin, an Ig-like C2 type domain is a site binding to neuropilin a1 and a2, and a basic rich region is a site exposing a portion capable of binding to neuropilin by being cleaved by furin. - In
FIG. 1(C) , VEGF165A includes a site capable of binding to VEGFR2 (KRD) which is an original receptor of VEGF and a heparin-binding domain (HBD) capable of binding to neuropilin. Further, in each of semaphorin and VEGF165A, an interface forming a dimer is present. - In
FIGS. 1(D) and (E), neuropilin broadly consists of five domains, and from the N-terminus, a1 and a2 domains are classified as CUB domains, and an Ig-like C2 type domain of semaphorin binds thereto. Particularly, this site forms a complex with plexin to increase binding capacity with semaphorin-plexin. The b1 and b2 domains are classified as FV/VIII domains, and the C-terminus of ligands of VEGF orclass 3 semaphorins bind thereto. Particularly, in this portion, a site to which heparin is capable of binding is present and this facilitates the binding of ligands with many (+) charged residues. Further, MAM induces oligomerization, trans-membrane domain (TM) enables neuropilin to be fixed onto cell surface, and in a cytosolic domain, a site capable of binding to a Postsynaptic density 95, Disk large, Zona occludens 1 (PDZ) domain is present. -
FIG. 2 is a schematic diagram illustrating a 2:2:2 complex including plexin of Sema3A and its co-receptor neuropilin. Sema3A and Sema3F present in nature form a homodimer, and the Sema domain interacts with the Sema domain of plexin. Further, the C-terminus of semaphorin interacts with b1 domain of neuropilin. -
FIG. 3 is a schematic diagram illustrating a binding complex of neuropilin, ligand, and TPP fused antibody heavy-chain constant region (Fc-TPP). It was expected that each protein at its C-terminus binds to neuropilin (particularly, to b1 domain) in the form of dimer. -
FIG. 4 is a schematic diagram illustrating a vector for expressing Fc-TPP in animal cells.FIG. 4(A) illustrates a part encoding Fc-TPP of a cleavage map of a vector for expression in host cells.FIG. 4(B) illustrates a whole map of a vector for expressing Fc-TPP in host cells. -
FIG. 5 is a schematic diagram illustrating TPP fused antibody heavy-chain constant region, and expression and purification SDS-PAGE. - In
FIG. 5(A) , the antibody heavy-chain constant region was prepared starting from a hinge at N-terminus to maintain two disulfide bonds and facilitate formation of a dimer. The external exposure degree of TPP was regulated using the peptide linker of four amino acids GAGA or fifteen amino acids (G4S)3 at the end of CH3, and thereafter 22 semaphorin derived sequences and improved derived sequences were added for preparation. - In
FIG. 5(B) , the formation of dimers of each clone on SDS-PAGE and purity upon purification can be confirmed. Also, the difference in size as much as the introduction of TPP can be confirmed. -
FIG. 6 illustrates a result of ELISA experiment for confirming the binding to b1b2 domains of NRP-1 and -2. VEGFR2-Fc, and b1b2 domains of NRP-1 and -2 were fixed onto plates, biotinylated peptides and Fc-TPP clones were bound at a concentration of 100 nM (in the case of peptides, 100 nM, 1 μM), and the binding was confirmed using anti-biotin antibody-alkaline phosphatase (AP) (SIGMA-ALDRICH co., USA) and pnitrophenyl palmitate (pNPP, SIGMA-ALDRICH co., USA).FIG. 6(A) illustrates a comparison of the control group with Sema3A derived peptide and Fc-TPP, andFIG. 6(B) illustrates a comparison of the control group with Sema3F derived peptide and Fc-TPP. By comparison, stronger bond to neuropilin1/2 occurred in A22p and F22p peptides and Fc-A22p/F22p clones inducing mutants, and slightly stronger bond occurred in clones using (G4S)3 linker among clones using GAGA and (G4S)3 linkers. No bond to the control group VEGFR2 was observed. Thereby, it was shown that TPP selectively binds to neuropilin. -
FIG. 7 confirms binding specificity of clones showing stronger binding capacity to neuropilin among Fc-TPP after confirming whether these clones are competitive with VEGF165A or Semaphorin3A to bind to neuropilin. It was confirmed that Fc-15A22p is competitive at a higher concentration of ligand than Fc-15F22p. -
FIG. 8 illustrates a result of FACS analysis of expression level of neuropilin-1 and -2 on the surface of cell lines used for biological identification of TPP in this experiment. As a result of conducting experiments in human prostatic cancer cell line (PPC-1), human head and neck cancer cell line (FaDu), human endothelial cell line (HUVEC), and human ovarian cancer cell line (SK-OV-3), it was confirmed that neuropilin-1 and neuropilin-2 were expressed in each cell line. -
FIG. 9(A) illustrates a result of FACS analysis in human ovarian cancer cell line (SK-OV-3) in order to confirm whether Fc-TPP binds to neuropilin-1 and -2 expressed on the cell surface. In the above biochemical identification result (FIG. 6 ), Fc, Fc-15A22P, and Fc-15F22P having binding capacity to b1b2 domains of neuropilin-1 and -2 were treated under the same conditions to confirm their binding capacities. As a result, it was confirmed that unlike the control group Fc, Fc-15A22P and Fc-15F22P bind to cell surface.FIG. 9(B) is a view illustrating a result confirming the binding capacity using the same manner as in the experiment ofFIG. 9(A) , after reacting purified b1b2 domains of neuropilin-1 and b1b2 domains of neuropilin-2 with each of Fc and Fc-TPP at room temperature in advance, in order to confirm that the binding capacity of Fc-TPP is specific to neuropilin-1 and -2 based on the result ofFIG. 9(A) . As a result, it was confirmed that the binding capacity of Fc-TPP was remarkably reduced in samples where b1b2 domains of neuropilin-1 and -2 in advance. This means that Fc-TPP binds specifically to neuropilin-1 and -2. -
FIGS. 9(C) and 9(D) illustrates a result of FACS analysis for comparing binding sites among VEGF165A, Sema3A, and Sema3F, which have binding capacity to neuropilin-1 and -2. As a result, it was confirmed that the binding capacity of Fc-TPP was inhibited by VEGF165, the binding capacity of Fc-15A22P by Sema3A, and the binding capacity of Fc-15F22P by Sema3F. -
FIG. 10 illustrates a result of observation of co-localization of Fc-TPP and neuropilin-1 and -2 through confocal microscopy analysis, in order to confirm the specific intracellular penetrating capacity of Fc-TPP into neuropilin. After treating human prostate cancer cell line PPC-1 with PBS, Fc, Fc-15A22P, and Fc-15F22P under the same conditions and staining the degree of infiltration into cells, it was observed that unlike Fc, Fc-15A22P and Fc-15F22P penetrated into cells. Further, it was confirmed that Fc-TPP and neuropilin-1 and -2 co-localize and thereby the penetrating capacity of Fc-TPP is specific to neuropilin-1 and -2. -
FIG. 11a illustrates a result of Western blot for identifying biological mechanism of Fc-TPP in HUVEC. Further, in order to confirm an effective format for TPP, single forms of peptide, A22, A22P, F22, and F22P were used, and for comparison depending on linker length with TPP, fusion types of Fc format, Fc-4A22, Fc-15A22, Fc-15A22P, Fc-4F22, Fc-15F22, and Fc-15F22P were used. The control group VEGF165A and Sema3A, unlike Sema3F, showed improvement of permeability in HUVEC, and this can be indirectly confirmed by a decrease in VE-cadherin. In the case of Sema3A derived TPP, Fc-15A22 and Fc-15A22P effectively decreased VE-cadherin, and VEGF165A and Sema3A showed the same result. Further, in the case of Sema3F derived TPP, the original ligand Sema3F did not induce a change in VE-cadherin, but Fc-15F22P decreased VE-cadherin under the same conditions. -
FIG. 11b illustrates a result of Transwell assay performed for confirming whether TPP improves permeable capacity of HUVEC. As a result, VEGF165, Sema3A, Fc-15A22, Fc-15A22P, and Fc-15F22P effectively improved permeability. On the other hand, single forms of peptide, A22, A22P, F22, and F22P did not improve permeability. This result is closely related to the result ofFIG. 11 a. -
FIG. 11c illustrates a result of immunohistochemistry for confirming TPP's infiltration capacity in actual tumor tissues. Human epidermoid carcinoma cell line A431 was transplanted into nude mice to confirm the effect of TPP through double staining with blood vessels (CD31). As a result, it was confirmed that unlike peptide A22P and the control group Fc, Fc-15A22 and Fc-15A22P selectively reached tumor cells, and effectively infiltrated into tumor cells. -
FIG. 11d illustrates a result of confirming the improvement of vascular permeability through Evans Blue assay. As a result, it was confirmed that unlike Fc, Fc-15A22P effectively improved tissue infiltration capacity, as shown in the above result. -
FIG. 11e illustrates a result of Western blot for a change in E-cadherin in human head and neck cancer cell line FaDu, in order to confirm the effect in cancer cell as well as vascular endothelial cell. As a result, in the case of Sema3A derived TPP, Fc-15A22 and Fc-15A22P induced a decrease in E-cadherin, unlike Sema3A, and in the case of Sema3F derived TPP, Fc-15F22P, like Sema3F, induced a decrease in E-cadherin. Further, upon reducing the expression of neuropilin-1 and -2 by co-injection of neuropilin-1 and -2 with siRNA, it was confirmed that Fc-TPP did not cause a decrease in E-cadherin, and thereby it was confirmed that Fc-TPP induced a decrease in E-cadherin specifically to neuropilin. -
FIG. 12a illustrates a result of tube formation assay performed for confirming whether Fc-15A22P inhibits tube formation of vascular endothelial cell by VEGF165A. As a result, it was confirmed that Fc-15A22P effectively inhibited the tube formation of endothelial cell induced by VEGF165A. -
FIG. 12b performed in vivo matrigel plug assay on mice, in order to identify whether Fc-15A22P can inhibit angiogenesis induced by VEGF165A. Also, the density of blood vessels was measured with anti-CD31 antibody through immunohistochemistry. As a result, it was confirmed that Fc-15A22P can inhibit angiogenesis induced by VEGF165A. -
FIG. 13(A) is a schematic diagram illustrating a fusion antibody (mAb-TPP) where a peptide binding to neuropilin is linked by a peptide linker to heavy-chain constant region C-terminus of single clone antibody. -
FIG. 13(B) is a schematic diagram illustrating that the fusion antibody of (A) binds to b1 domain of neuropilin-1 or -2. -
FIG. 13(C) is a schematic diagram illustrating the mechanism expected when the above fusion antibody (mAb-TPP) is introduced into the body. The binding of peptide fused antibody to neuropilin-1 or -2 overexpressed in tumor vascular endothelium and various tumor cells increases selective distribution of tumor tissue, increases extravasation, and induces signal transduction increasing infiltration into tumor cells, which may result in remarkably increasing specific distribution of tumor tissue of fusion antibody and infiltration thereinto. -
FIG. 14 is an example of a cleavage map of a vector for expressing IgG heavy chain-TPP.FIG. 14(A) represents a part coding IgG heavy chain-TPP of map of a vector for expression in host cells.FIG. 14(B) represents a whole map of a vector for expressing IgG heavy chain-TPP in host cells. -
FIG. 15 is an example of a cleavage map of a vector for expressing IgG light chain in animal cells.FIG. 15(A) represents a part coding IgG light chain of map of a vector for expression in host cells.FIG. 15(B) represents a whole map of a vector for expressing IgG light chain in host cells. -
FIG. 16(A) is a schematic diagram illustrating an antibody constructed by introducing TPP into Fc C-terminus of Cetuximab IgG which is the existing anti-EGFR IgG.FIG. 16(B) illustrates a result of analyzing the size and purity on SDS-PAGE under reducing and non-reducing conditions, after transient expression and purification in HEK293K cells through co-transformation.FIG. 16(C) illustrates a result confirming through Sandwich ELISA that Cetuximab-TPP is capable of binding to antigen EGFR and neuropilin simultaneously. -
FIG. 17(A) is a schematic diagram illustrating an antibody introducing TPP into the existing anti-HER2 IgG Trastuzumab in the same manner as the experiment above.FIG. 17(B) illustrates a result of analyzing the size and purity on SDS-PAGE under reducing and non-reducing conditions, after transient expression and purification in HEK293F cells through co-transformation.FIG. 17(C) illustrates a result confirming through Sandwich ELISA that Trastuzumab-TPP is capable of binding to antigen Her2 and neuropilin simultaneously. -
FIG. 18 illustrates a result of Surface Plasmon Resonance (SPR) for confirming whether binding capacity between mAb and mAb-TPP depending on pH for FcRn is similar to each other. It was proven that mAb (Cetuximab, Trastuzumab) showed a binding curve at pH 6.0, and mAb-TPP (Cetuximab-TPP, Trastuzumab-TPP) maintained the properties of wild-type antibodies which do not completely bind at pH 7.4. -
FIG. 19a illustrates a result of IHC for confirming the infiltration capacity of Cetuximab-TPP in tumor tissues. After transplanting into nude mice each of human head and neck cancer cell line FaDu and human epidermoid carcinoma A431 in which EGFR is expressed, Cetuximab and Cetuximab-15A22P were injected thereto to confirm infiltration capacity into tissue through double staining with blood vessels (CD31). As a result, it was confirmed that Cetuximab infiltrated into the periphery of blood vessels, whereas Cetuximab-15A22P infiltrated into the tissue to be further far away from blood vessels (upper panel). This was quantified using Image J (lower panel). -
FIG. 19b illustrates a result of Western blot performed from cancer cell tissues extracted from mice under the conditions ofFIG. 18 b. -
FIG. 19c illustrates a result of experiment on inhibition of cancer cell growth in nude mice for confirming whether improved infiltration into tumor tissue actually affects inhibition of cancer cell. As a result, it was confirmed that Cetuximab and Cetuximab-15A22P inhibited the growth of cancer cell, compared with PBS, and that Cetuximab-15A22P more effectively inhibited the growth of cancer cell under the same conditions. -
FIG. 19d illustrates a result of measuring the weights of mice at the time of conducting the above experiment. When compared with the experimental group into which Cetuximab was injected, the experimental group mice into which Cetuximab-15A22P was injected had no difference in weight. This indirectly proves that Cetuximab-15A22P does not have any toxicity against mice, compared with Cetuximab. -
FIG. 20a illustrates a result of IHC for confirming the infiltration capacity of Trastuzumab-TPP in tumor tissues. After injecting to nude mice each of human ovarian cancer cell line SK-OV-3 in which HER2 is expressed, Trastuzumab and Trastuzumab-15A22P were injected thereto to confirm infiltration capacity into tissue through double staining with blood vessels (CD31). As a result, it was confirmed that Trastuzumab infiltrated into the periphery of blood vessels, whereas Trastuzumab-15A22P infiltrates within the tissue to be further away from blood vessels (upper panel). This was quantified using Image J (lower panel). -
FIG. 20b illustrates a result of Western blot performed from cancer cell tissues extracted from mice under the conditions ofFIG. 19 a. -
FIG. 20c illustrates a result of experiments for confirming the effect of Trastuzumab-TPP on inhibiting the growth of cancer cell. As a result, it was confirmed that under the same conditions, Trastuzumab-15A22P more effectively induced inhibition on the growth of cancer cell than Trastuzumab. -
FIG. 20d illustrates a result of measuring weights of mice during the above experiments. When compared with mice of the experimental group into which Trastuzumab was injected, mice of the experimental group into which Trastuzumab-15A22P was injected had no difference in weights. This indirectly proved that Trastuzumab-15A22P did not have any toxicity against mice, when compared with Trastuzumab. -
FIG. 21 illustrates a result of in vitro evaluation on the degree of cell growth inhibition by treating human head and neck cancer cell line FaDu and human ovarian cancer cell line SK-OV-3 with Fc-TPP and mAb-TPP. As a result, Cetuximab and Cetuximab-15A22P inhibited cell growth by about 30% in cell line FaDu, and Trastuzumab and Trastuzumab-15A22P inhibited cell growth by about 30-35% in cell line SK-OV-3. It was confirmed that there was no difference between mAb and mAb-TPP. Likewise, Fc and Fc-TPP did not show an effect of inhibiting cell growth in both FaDu and SK-OV-3. The mechanism of TPP by neuropilin means that TPP itself maximizes the effect by increasing infiltration into tumor tissue without directly affecting the growth of cancer cells. - Hereinafter, the present invention will be described in more detail with examples. However, these examples are provided for illustrative purposes only, and are not intended to limit the scope of the present invention.
- In order to induce peptides specifically binding to neuropilin, sequences of ligands known to bind to neuropilin, i.e., vascular endothelial cell growth factor (VEGF-A, B, C, D), semaphorin 3-A, B, C, D, E, F (
class 3 semaphorin), fibroblast growth factor-2 (FGF2), hepatocyte growth factor (HGF), and galectin-1 were analyzed. - Representatively, the whole sequences of VEGF-A and
class 3 semaphorin were selected from the PubMed Entrez Protein Database. - As illustrated in
FIGS. 1(A) and 1(B) , secretedclass 3 semaphorins (Sema3s) were identified fromsemaphorins 3A to 3G, and they have three domains in common. Upon investigation from the N-terminus, a sema domain is a site binding to plexin, an Ig-like C2 type domain is a site binding to neuropilin a1 and a2, and a basic rich region is a site exposing a portion capable of binding to neuropilin by being cleaved by furin. - As illustrated in
FIG. 1(C) , VEGF-A, which most strongly binds to neuropilin, among ligands of neuropilin known until now, particularly, VEGF165A, is divided into a site capable of binding to a primary receptor of VEGF, VEGFR2 and a heparin-binding domain (HBD) capable of binding to neuropilin. When binding to neuropilin-1, VEGF165A contains a portion (115-159, Exon7a, 7b) where there are many (+) charged residues binding to heparin which binds to neuropilin-1, in addition to the portion (160-165, Exon8a) binding to neuropilin-1. The portion where there are many (+) charged residues may cause a non-specific binding to heparin which resides in outer walls of cells. Also, VEGF165A contains cysteine at a portion corresponding to Exon8, and thus is expected to cause a decrease in an amount of expression, when VEGF165A is fused with a protein such as an antibody and expressed, and thus it is excluded. - As illustrated in
FIGS. 1(D) and 1(E) , neuropilin broadly consists of five domains, and from the N-terminus, a1 and a2 domains are classified as CUB domains, and an Ig-like C2 type of semaphorin binds thereto. Particularly, this site forms a complex with plexin, and plays a role of increasing the binding force with semaphorin-plexin. The b1 and b2 domains are classified as FV/VIII domains, and the C-terminus of VEGF family ligand orclass 3 semaphorin ligand binds thereto. Particularly, in this portion, a site to which heparin is capable of binding is present and this facilitates the binding of ligands with many (+) charged residues. Further, MAM induces oligomerization, trans-membrane domain (TM) enables neuropilin to be fixed onto cell surface, and in a cytosolic domain, a site capable of binding to a Postsynaptic density 95, Disk large, Zona occludens 1 (PDZ) domain is present. -
FIG. 2 is a schematic diagram illustrating a 2:2:2 complex including plexin of Sema3A and its co-receptor neuropilin. Sema3A and Sema3F present in nature form a homodimer, and the Sema domain interacts with the Sema domain of plexin. Further, the C-terminus of semaphorin interacts with b1 domain of neuropilin. -
FIG. 3 is a schematic diagram illustrating a binding complex of neuropilin, ligand, and TPP fused antibody heavy-chain constant region (Fc-TPP). It was expected that similar to Sema3A and Sema3F present in nature, each protein at its C-terminus binds to neuropilin (particularly, to b1 domain) in the form of dimer. - As a result of sequence similarity analysis based on the structures and sequences of VEGF-A and
class 3 semaphorin as above, it was found that there is a similarity in their C-terminus Particularly, the binding ofclass 3 semaphorin to neuropilin is known to have activity after cleavage by furin, and here the similarity in the C-terminus of VEGF-A which interacts with neuropilin was found. This is shown in Table 1 below. The following Table 1 shows the result of amino acid sequence similarity analysis of C-terminal portions ofclass 3 semaphorins and VEGF-A family ligands which are ligands of neuropilin-1 and -2. -
TABLE 1 Sema3A CEQVWKRDRKQRRQRPGHTFGNSNKWKHLQENKKGRNRR (722-760) Sema3B CRP-----QPALQSL--PLE----------SRRKGR N RR (710-731) Sema3C CKDTRQQRQQGDESQ--KMRGDYGKLKALINSRKSR N RR (709-745) Sema3D CEQMWRREK--RRQR---NKGGP-KWKRMQEMKKKR N RR (731-763) Sema3E CTDRKRKKLKMSPSK--WKYANPQEKKLRSKPERYR L PR (734-770) Sema3F CQGYWRHVPPSPREA----PGAP-RSPEPQDQKKPR N RR (756-779) Sema3G CFRSRSRGKQARGKS----------WA---GLELGK K MK (750-774) VEGF165 CKNTDSRCKARQLEL------------NERTCRCDK P RR (138-165) VEGF145 ARQEKKSVRGRGRGQ----RRRRKRSRYKSWSVCDK P RR (110-145) Clustal . : : Consensus - Based on the above sequence similarity, potential sequences are induced for introducing into an antibody from C-terminus to N-terminus of the portion where
class 3 semaphorins are cleaved by furin. The following Table 2 shows the amino acid sequence information of potential binding sites ofclass 3 semaphorins and VEGF-A family ligands to neuropilin. -
TABLE 2 Nrp1/2 ligand C-terminus sequence Sema3A EQVWKRDRKQRRQRPGHTPGNSNKWKHLQENKKGRNRR Sema3B RPQPALQSLPLESRRKGRNRR Sema3C KDTRQQHQQGDESQKMRGDYGKLKALINSRKSRNRR Sema3D EQMWHREKRRQRNKGGPKWKHMQEMKKKRNRP Sema3E TDRKRKKLKMSPSKWKYANPQEKKLRSKPEHYRLPR Sema3F QGYWRHYVPPSPREAPGAPRSPEPQDQKKPRNRR VEGF165 ARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR VEGF145 ARQEKKSVRGKGKGQKRKRKKSRYKSWSVCDKPRR VEGF121 ARQEKCDKPRR - 22 amino acids were selected from the C-terminus sequence information of each of Sema3A and Sema3F of which binding capacity to neuropilin is well known. Specifically, A22 peptide having 22 amino acids derived from residues 739-760 which are part of the basic domain of Sema3A and F22 peptide having 22 amino acids derived from residues 758-779 which are part of the basic domain of Sema3F were selected.
- Also, as a result of comparing amino acid sequences of potential neuropilin binding sites between
class 3 semaphorins and VEGF-A family ligands, it was found thatclass 3 semaphorins preserved the third amino acid residue from C-terminus as asparagine (Asn), whereas VEGF-A family ligands which have high affinity with neuropilin preserved the residue as proline (Pro) (Table 2). Particularly, it was expected that the proline plays a role in limiting a domain to a specific structure in neuropilin interactions and greatly contributes to affinity between neuropilin-domains. In order to design a peptide with improved affinity with neuropilin using the same logic, A22p and F22p where the last third amino acids of A22 and F22 are replaced with proline were designed. The following Table 3 shows peptide sequences and SEQ ID NO. of A22 and F22 having amino acid sequences derived from C-terminus of Sema3A and Sema3F binding to neuropilin, and of A22p and F22p designed to increase affinity with neuropilin. The underlines are used to highlight the parts inducing mutants. -
TABLE 3 Neuropilin SEQ TPP Ligand target ID Name derived sequence NO. A22 Sema3A HTPGNSNKWKH SEQ ID LQENKKGRNRR NO: 1 A22p Sema3A HTPGNSNKWKH SEQ ID LQENKKGRPRR NO: 2 F22 Sema3F REAPGAPRSPE SEQ ID PQDQKKPRNRR NO: 3 F22p Sema3F REAPGAPRSPE SEQ ID PQDQKKPRPRR NO: 4 - In order to perform experiments on the length of linkers for introducing four types of peptide sequences designed in the above Example 1 into C-terminus of a constant region Fc of a human antibody IgG1, linkers having 4 amino acids consisting of glycine, serine, and alanine, or 15 amino acids were selected. The selected linkers have sequences of GAGA and (GGGGS)3. The clone names and sequence information are shown in the following Table 4.
-
TABLE 4 Amino acid sequence fused to C-terminal of Fc Length (from N-terminus of to C-terminus) entire TPP Linker Neuropilin amino SEQ ID Name sequence target sequence acid NO. 4A22 GAGA HTPGNSNEWKHLQEN 26 SEQ ID KKGRNRR (SEQ ID NO: 5 NO: 1) 15A22 GGGGSGGG HTPGNSNEWKHLQEN 37 SEQ ID GSGGGGS KKGRNRR (SEQ ID NO: 6 NO: 1) 15A22p GGGGSGGG HTPGNSNKWKHLQEN 37 SEQ ID GSGGGGS KKGRPRR (SEQ ID NO: 7 NO: 2) 4F22 GAGA REAPGAPRSPEPQDQ 26 SEQ ID KKPRNRR (SEQ ID NO: 8 NO: 3) 13F22 GGGGSGGG REAPGAPRSPEPQDQ 37 SEQ ID GSGGGGS KKPRNRR (SEQ ID NO: 9 NO: 3) 15F22p GGGGSGGG REAPGAPRSPEPQDQ 37 SEQ ID GSGGGGS KKPRPRR (SEQ ID NO: 10 NO: 4)
Names and sequence information of TTPs and linkers linking to C-terminus of antibody heavy chain regions - Peptides (SEQ ID NOS: 5-10) shown in Table 4 were fused to C-terminus of Fc, a constant region of human antibody IgG1, to have the properties of binding neuropilin in a bivalent form. This is designed to activate target neuropilin receptors, by copying Sema3A and Sema3F ligands which bind to neuropilin as homodimer.
- A Fc-TPP expression vector where the designed TPP is fused to C-terminus of Fc was cloned in an expression vector for animal cells to be implemented.
FIG. 4(A) illustrates a part coding Fc-TPP of a cleavage map of a vector for expression in host cells.FIG. 4(B) illustrates a whole map of a vector for expressing Fc-TPP in host cells. - Before preparing Fc-TPP, Fc was cloned into the pSecTag2A vector with the restriction enzyme AscI/HindIII. Each Fc-TPP was substituted with the restriction enzyme BsrGI/HindIII from CH3 till the terminus by performing PCR with same forward primers starting from CH3 having Fc as a template and their respective reverse primers for introducing TPP into C-terminus.
- When Fc and Fc-TPP are expressed as above, they show different sizes as much as those of introducing TPP to purified proteins on SDS-PAGE (
FIG. 5(B) ) Fc was used as a control group for the size. Biotin-peptide of sequence derived from semaphorin consisting of 22 amino acids which was used as a control group was prepared by chemical synthesis (Peptron, Korea). - Proteins were expressed using transient transfection of plasmid (
FIG. 4 ) encoding each Fc-TPP constructed in Example 2 above, with HEK293-F system (Invitrogen). In a shake flask, HEK293-F cells (Invitrogen) which were suspended and grown in the serum-free FreeStyle 293 expression medium (Invitrogen) were transfected with a mixture of plasmid and polyethylenimine (PEI) (Polyscience). When transfecting 200 mL in the shake flask (Corning), HEK293-F cells were seeded in 100 ml medium at a density of 2.0E*6 cells/ml and incubated at 120 rpm, 8% CO2. Thereafter, plasmid encoding Fc-TPP was diluted in 10 ml FreeStyle 293 expression medium (Invitrogen) to be 250 μg (2.5 μg/ml), and mixed with 10 ml medium where PEI 750 μg (7.5 μg/ml) was diluted to be reacted at room temperature for 10 minutes. Then, the reacted mixture medium was put into the 100 ml seeded cells and incubated at 120 rpm, 8% CO2 for 4 hours, and then the other 100 ml FreeStyle 293 expression medium was added thereto, followed by incubation for 7 days. The supernatant was collected after 7 days. - Proteins were purified from the collected cell culture supernatant by referring to the standard protocol. Antibodies applied to Protein A Sepharose column (GE healthcare) and were washed with PBS (pH 7.4). The antibodies were eluted at pH 3.0 using 0.1 M glycine buffer, and thereafter samples were immediately neutralized using 1 M Tris buffer. The buffer was changed to PBS (pH 7.4) using Pierce Dextran Desalting Column (5K MWCO). Thereafter, the eluted antibody fragments were concentrated using the centrifugal concentrator MILLIPORE Amicon Ultra (10 MWCO), and purified Fc-TPP was quantified using absorbance and absorption coefficient at wavelength 280 nm. The purified Fc-TPP was analyzed on SDS-PAGE under reducing and non-reducing conditions.
-
FIG. 5(A) is a schematic diagram illustrating TPP fused antibody heavy chain constant region. The antibody heavy-chain constant region was prepared starting from a hinge at the N-terminus to maintain two disulfide bonds and facilitate formation of a dimer. The external exposure degree of TPP was regulated using the peptide linker of GAGA or (GGGGS)3 at the end of CH3, and thereafter 22 semaphorin derived sequences were added for preparation. -
FIG. 5(B) illustrates a result of analysis of SDS-PAGE of the purified Fc-TPP under reducing and non-reducing conditions. FromFIG. 5(B) , the formation and purity of dimmers of each clone on SDS-PAGE can be confirmed. - The following Table 5 shows the yields of proteins produced per 1 L medium of the purified TPP fused protein. The results obtained from three times experiments were statistics processed, and ± represents standard deviation. The obtained yields of proteins are not remarkably different from those of wild-type proteins.
-
TABLE 5 Name of clone Yield (mg/l) Name of clone Yield (mg/l) Fc 34.2 ± 4.8 Fc-4F22 34.8 ± 2.1 Fc-4A22 24.3 ± 4.7 Fc-15F22 35.3 ± 9.8 Fc-15A22 45.0 ± 8.3 Fc-15F22p 35.7 ± 5.8 Fc-15A22p 42.1 ± 8.6
Comparison of Fc-TPP expression purification yields - The binding capacity of purified Fc-TPP to b1b2 domains of neuropilin-1 and -2 (neuropilin (NRP)1/2-b1b1 domain) was confirmed in Enzyme Linked Immunosorbent Assay (ELISA).
- As the control group, VEGF165A, Sema3A (26-760), Sema3F (19-779) and Fc, and their respective Fc-TPPs were biotinylated using the NHS-biotin kit (SIGMA-ALDRICH co., USA).
- The target molecules, b1b2 domains (273-586) of neuropilin-1 and b1b2 domains (275-595) of neuropilin-2, and the control group VEGFR2 (46-753) were bound in an amount of 1 μg each in 96-well EIA/RIA plates (COSTAR Corning In., USA) at room temperature for 1 hour, and then washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA). After binding for 1 hour with 5% skim milk (pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA), it was washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA). For the control group, biotinylated VEGF165A, Sema3A, Sema3F and Fc, and for the experimental group, their respective Fc-TPPs and TPP peptides, were washed three times for 10 minutes with 100 nM (or were bound in a concentration of 1 μM of peptide and thereafter washed three times for 10 minutes with 0.1% PBST). After binding to alkaline phosphatase (AP)-conjugated anti-biotin mAb (Sigma, USA) and reacting with pnitrophenyl palmitate (pNPP, SIGMA-ALDRICH co., USA), absorbance at 405 nm was quantified. From the ELISA result obtained from 30 minute-reaction of AP-pNPP, the binding capacity of expressed and purified Fc-TPP to b1b2 domains of neuropilin-1 and -2 was confirmed.
-
FIG. 6 illustrates a result of ELISA experiments for confirming the binding to b1b2 domains of neuropilin-1 and -2.FIG. 6(A) illustrates a comparison of the control group with Sema3A derived peptide and Fc-TPP, andFIG. 6(B) illustrates a comparison of the control group with Sema3F derived peptide and Fc-TPP. By comparison, stronger bond occurred in A22p and F22p peptides and Fc-A22p/F22p clones relatively inducing mutants, and slightly stronger bond occurred in clones using (G4S)3 linker among clones using GAGA and (G4S)3 linkers. - In order to confirm the binding specificity of Fc-TPP to b1b2 domains of neuropilin-1 and -2, ELISA for binding competition of the control group VEGF165A and Sema3A was performed.
- Specifically, b1b2 domains (273-586) of neuropilin-1 and b1b2 domains (275-595) of neuropilin-2 were bound in 96-well EIA/RIA plates (COSTAR Corning In., USA) at room temperature for 1 hour, and then washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA). After binding for 1 hour with 5% skim milk (pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA), it was washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA). Mixtures where Fc-15A22p (30 nM) and Fc-15F22p (30 nM) showing high binding force were mixed with VEGF165A (25 nM to 0.02 nM) and Sema3A (3.3 μM to 0.2 nM) per concentration were produced to bind to b1b2 domains of neuropilin-1 and -2. After binding to alkaline phosphatase (AP)-conjugated anti-biotin mAb (Sigma, USA) and reacting with pnitrophenyl palmitate (pNPP, SIGMA-ALDRICH co., USA), absorbance at 405 nm was quantified. From the ELISA result, it was confirmed that VEGF165A, Sema3A and Fc-TPP are competitive to bind to b1b2 domains of neuropilin-1 and -2.
-
FIG. 7 illustrates a result of confirmation whether clones showing stronger binding force to neuropilin among Fc-TPP are competitive with VEGF165A and Sema3A to bind to neuropilin. As illustrated inFIG. 7 , it was confirmed that Fc-15A22p is competitive at a higher concentration of ligand than Fc-15F22p. - In order to more specifically confirm the binding force of Fc-TPP to b1b2 domains of neuropilin-1 and -2, surface plasmon resonance (SPR) was performed. Biacore2000 (GE healthcare) was used, and the binding force of VEGF165, Sema3F, Sema3A, Fc-4A22, Fc-4F22, Fc-15A22, Fc-15F22, Fc-15A22p and Fc-15F22p, of which binding capacity was confirmed by ELISA, to b1b2 domains of neuropilin-1 and -2 were analyzed.
- Specifically, each b1b2 domain of neuropilin-1 and -2 was diluted with 10 mM Na-acetate buffer (pH 4.0), and fixed onto CM5 sensor chips (GE healthcare, USA) in about 1000 response units (RU). HBS-EP buffer (10 mM Hepes, 3 mM ethylenediaminetetraacetic acid, and 0.005% surfactant P20 (pH 7.4), GE Healthcare) was analyzed at a
stream velocity 30 μl/min, and VEGF165 at a concentration of 80 nM to 5 nM, Sema3F and Sema3A at a concentration of 1 μM to 62.5 nM, and Fc-TPP at a concentration of 25 μM to 1.5625 μM were analyzed. After bond and dissociation analysis, the regeneration of CM5 chip was performed by streaming a buffer (20 Mm NaOH, 1 M NaCl, pH 10.0) at astream velocity 30 μl/min for 1 minute. Each sensorgram obtained by 3 minute-bond and 3 minute-dissociation was normalized and subtracted by comparing with blank cells to calculate the binding affinity. - Table 6 shows a result of the binding affinity of Sema3A derived A22 peptide and a single mutant A22p in the form of Fc-TPP with b1b2 domains of neuropilin-1 and -2 (
NRP 1 and 2), using surface plasmon resonance (SPR,BIACORE 2000, GE healthcare, USA). Table 7 shows a result of the binding affinity of Sema3F derived F22 peptide and a single mutant F22p in the form of Fc-TPP with b1b2 domains of neuropilin-1 and -2. - As shown in Tables 6 and 7, the difference in binding affinity between clones having GAGA linker and clones having (GGGGS)3 linker is about 10 times, when compared with Fc-4A22 and Fc-15A22. Pro mutant clones show the binding affinity about 100 times as high as those fused with wild-type peptide binding to neuropilin. At least 5 sensorgrams were used for analysis, and results obtained from twice experiments were statistics processed. ± represents standard deviation of results of individual experiments.
-
TABLE 6 Association rate (Dissociation rate Binding affinity Clone Receptors ka (M−1s−1) kd (s−1) KD(M) VEGF165 NRP1-b1b2 9.75 ± 0.53 × 105 3.42 ± 0.29 × 10−3 3.51 ± 0.36 × 10−9 VEGF165 NRP2-b1b2 1.17 ± 0.12 × 105 3.73 ± 0.19 × 10−3 3.20 ± 0.14 × 10−8 Sema3A NRP1-b1b2 2.89 ± 0.07 × 104 8.08 ± 0.06 × 10−4 2.79 ± 0.14 × 10−8 Sema3A NRP2-b1b2 6.96 ± 0.03 × 103 1.61 ± 0.28 × 10−3 2.31 ± 0.22 × 10−7 Fc-4A22 NRP1-b1b2 1.02 ± 0.14 × 103 1.93 ± 0.05 × 10−2 1.80 ± 0.14 × 10−5 Fc-4A22 NRP2-b1b2 6.09 ± 0.63 × 103 1.23 ± 0.16 × 10−1 2.02 ± 1.41 × 10−5 Fc-15A22 NRP1-b1b2 4.86 ± 0.98 × 103 2.95 ± 0.04 × 10−2 6.06 ± 0.42 × 10−6 Fc-15A22 NRP2-b1b2 3.94 ± 0.42 × 103 3.87 ± 0.09 × 10−2 9.8 ± 0.14 × 10−6 Fc-15A22p NRP1-b1b2 9.15 ± 0.06 × 103 5.77 ± 0.16 × 10−4 6.3 ± 0.21 × 10−8 Fc-15A22p NRP2-b1b2 6.06 ± 0.04 × 103 3.76 ± 0.17 × 10−4 6.2 ± 0.14 × 10−8 -
TABLE 7 Association rate Dissociation rate Binding affinity Clone Receptors ka (M−1s−1) kd (s−1) KD(M) VEGF165 NRP1-b1b2 9.75 ± 0.53 × 105 3.42 ± 0.29 × 10−3 3.51 ± 0.36 × 10−9 NRP2-b1b2 1.17 ± 0.12 × 105 3.73 ± 0.19 × 10−3 3.20 ± 0.14 × 10−8 Sema3F NRP1-b1b2 3.98 ± 0.69 × 103 9.73 ± 0.19 × 10−4 2.45 ± 0.14 × 10−7 NRP2-b1b2 4.34 ± 0.24 × 103 1.50 ± 0.35 × 10−3 3.45 ± 0.32 × 10−7 Fc-4F22 NRP1-b1b2 2.18 ± 0.12 × 102 2.25 ± 0.18 × 10−3 1.03 ± 0.04 × 10−5 NRP2-b1b2 1.21 ± 0.15 × 102 1.57 ± 0.40 × 10−3 1.30 ± 0.21 × 10−5 Fc-15F22 NRP1-b1b2 1.35 ± 0.24 × 103 5.34 ± 0.24 × 10−2 3.97 ± 0.31 × 10−5 NRP2-b1b2 1.05 ± 0.03 × 103 1.30 ± 0.21 × 10−2 1.24 ± 0.18 × 10−5 Fc-15F22p NRP1-b1b2 1.73 ± 0.26 × 104 1.38 ± 0.12 × 10−3 7.97 ± 0.51 × 10−8 NRP2-b1b2 1.34 ± 0.24 × 104 1.23 ± 0.16 × 10−3 9.24 ± 0.17 × 10−8 - First of all, in order to confirm the expression level of neuropilin-1 and -2 on the surface of cell lines used in the experiments for biological identification of TPP, FACS analysis was performed.
-
FIG. 8 is a view illustrating that neuropilin-1 and -2 are expressed in cell lines. As illustrated, as a result of conducting experiments in human prostatic cancer cell line (PPC-1), human head and neck cancer cell line (FaDu), human endothelial cell line (HUVEC), and human ovarian cancer cell line (SK-OV-3), it was confirmed that neuropilin-1 and neuropilin-2 were expressed in each cell line. - In order to confirm whether Fc-TPP binds to neuropilin-1 and -2 expressed on cell surface, FACS analysis was performed, and Fc-15A22P and Fc-15F22P which were confirmed to specifically bind to neuropilin-1 and -2 were used.
- Specifically, after incubating human ovarian cancer cell line (SK-OV-3) under conditions of 5% CO2 and 37° C., 1×105 cells per sample were resuspended in a PBS buffer supplemented with 2% BSA and transferred to a FACS tube for conducting experiments. Each of Fc, Fc-15A22P, and Fc-15F22P were diluted with the buffer to be 1 μM and reacted at 4° C. for 1 hour, and then cellular binding proteins were stained using FITC-conjugated antibodies (Sigma) recognizing Fc for analysis with FACS Calibur (BD Bioscience).
-
FIG. 9 illustrates a result of FACS analysis in human prostate cancer cell line (PPC-1) in order to confirm whether Fc-TPP binds to neuropilin-1 and -2 expressed on the cell surface. As illustrated inFIG. 9(A) , it was confirmed that unlike Fc, Fc-15A22P and Fc-15F22P bind to the surface of PPC-1 cells. - Also, in order to confirm that such binding capacity is specific to neuropilin-1 and -2, 2 μM of b1b2 domains of neuropilin-1 and -2 which were used in the above experiments were mixed with each of 1 μM of Fc, Fc-15A22P, and Fc-15F22P and reacted at room temperature for 20 minutes. Then, the mixture was reacted with cell line SK-OV-3 at 4° C. for 1 hour in the same manner and proteins were stained for conducting FACS analysis. As illustrated in
FIG. 9(B) , it was confirmed that the binding capacity to Fc-TPP was remarkably reduced in samples where Fc-15A22P and Fc-15F22P were reacted in advance with b1b2 domains of neuropilin-1 and -2. This means that Fc-TPP binds specifically to neuropilin-1 and -2. - Additionally, in order to confirm the site where Fc-TPP binds to neuropilin-1 and -2, each of Fc-15A22P and Fc-15F22P was mixed with VEGF165A (1 μg/ml), and the binding capacity was confirmed in the same manner as above. As illustrated in
FIG. 9(C) , it was confirmed that the binding capacity was reduced by VEGF165A, and this means competitive binding with VEGF165A. - Also, Fc-15A22P and Fc-15F22P were mixed with Sema3A and Sema3F (100 μg/ml), respectively, which are ligands from which Fc-15A22P and Fc-15F22P were derived, and reacted, to confirm the binding capacity. As illustrated in
FIG. 9(D) , the binding capacity of each of Fc-15A22P and Fc-15F22P was reduced by Sema3A and Sema3F. This means that the binding of Fc-TPP to neuropilin-1 and -2 is specific similar to the original ligands. - In order to confirm whether Fc-TPP has intracellular penetrating capacity by neuropilin-1 and -2, like other neuropilin ligands having intracellular penetrating capacity, confocal microscopy was used to observe intracellular penetration and co-localization with neuropilin-1 and -2.
- Specifically, 5×104 PPC-1 cells were plated into 24-well plates containing DMEM medium 0.5 ml supplemented with 10% FBS per well, followed by incubation at 37° C. for 24 hours at 5% CO2. When the cells were stabilized, each well was washed with PBS 0.5 ml. Then, Fc, Fc-15A22P, and Fc-15F22P were diluted with 0.5 ml of transfection optimized medium (TOM, WelGENE Inc., Korea) to be 1 μM, followed by incubation at 37° C. for 1 hour under 5% CO2 condition. After the medium was removed and each well was washed with PBS, Fc-TPP was stained with an antibody (Sigma) which specifically recognizes the FITC (green fluorescence)-conjugated Fc, and neuropilin-1 and -2 was stained with a primary antibody (SantaCruz) which recognizes each of them and with a TRITC (red fluorescence)-conjugated secondary antibody (Sigma). Nuclei were stained (blue fluorescence) with DAPI and observed under confocal microscopy.
-
FIG. 10 illustrates a result of observation of co-localization of Fc-TPP and neuropilin-1 and -2 through confocal microscopy analysis, in order to confirm the specific intracellular penetrating capacity of Fc-TPP through neuropilin. As illustrated inFIG. 10 , it was confirmed that the control group Fc did not penetrate into cells, whereas Fc-15A22P and Fc-15F22P penetrated into cells and were co-localized with neuropilin-1 and -2, respectively. This means that Fc-TPP has specific penetrating capacity by neuropilin-1 and -2. - Sema3A or VEGF165A is known to increase vascular permeability using neuropilin-1 (NRP1) as a co-receptor. During this process, a decrease in vascular endothelial (VE)-cadherin, phosphorylation, etc. of endothelial cells occur. That is, VE-cadherin or epithelial (E)-cadherin involves in adhesion between endothelial cells, while forming the base of adherent junction between endothelial cells and epithelial cells. The change in molecules relieve the density of adherent junction, which results in increasing permeability of blood vessels and improving intracellular permeability.
- As an experimental method for indirectly confirming improvement of vascular permeability based thereon, a change in VE-cadherin was confirmed through a Western blot. Specifically, HUVECs were seeded into 6-well plates at a density of 3×105 per well, followed by incubation for 24 hours. Thereafter, single peptides and Fc-TPP were treated with 1 μM for 10 minutes to perform a Western blot. Gel subjected to SDS-PAGE was transported into PVDF membrane, a primary antibody (SantaCruz) and an HRP-conjugated secondary antibody (SantaCruz) which recognize VE-cadherin and β-actin, respectively were used for detection. ImageQuant LAS4000 mini (GE Healthcare) was used for analysis.
-
FIG. 11a illustrates a result of Western blot for identifying biological mechanism of Fc-TPP in HUVEC. As illustrated inFIG. 11a , in the case of Sema3A derived protein, when treating the control group VEGF165A and Sema3A, a decrease in VE-cadherin was observed. In the case of single peptides A22 and A22P, no change in VE-cadherin was observed. Also, in the case of Fc and Fc-4A22, VE-cadherin was not decreased, whereas in the case of Fc-15A22 and Fc-15A22P, VE-cadherin was decreased (left panel). In the case of Sema3F derived TPP, only the control group VEGF165A and Fc-15F22P induced a decrease in VE-cadherin (right panel). - Based on the above experimental results, as experiments for confirming the improvement of vascular endothelial cell permeability, single peptides and Fc-TPP were treated to conduct Transwell assay.
- Specifically, HUVECs were seeded into Transwell plate (Corning) at a density of 5×104 per well in a upper chamber using Endothelial Growth Medium, PromoCell (EGM) medium, followed by incubation at 37° C. for 3 days under 5% CO2 condition. Thereafter, after changing the medium to Endothelial Basal Medium (EBM, PromoCell), the control group VEGF165A, Sema3A and Sema3F were treated with about 1.3 nM and single peptides and Fc-TPP with 1 μM for 30 minutes. Thereafter, Dextran-FITC (Sigma) 50 μg was put into the upper chamber and the medium of the lower chamber was sampled to measure its fluorescence after 30 minutes, using the principle that a fluorescent substance is observed in the lower chamber when the permeability in HUVECs was improved.
-
FIG. 11b illustrates a result of Transwell assay performed for confirming whether TPP improves permeability of HUVEC. As illustrated inFIG. 11b , it was confirmed that in the case of VEGF165A andSema 3A, permeability of HUVEC was improved, and in the case of single peptides A22 and A22P, and Fc-4A22 with short Fc part and linker of TPP, the improvement of vascular permeability was not induced. On the other hand, in the case of fusion forms Fc-15A22 and Fc-15A22P, it was confirmed that permeability of HUVEC was improved. In the case of Sema3F derived TPP, HUVEC permeability was not improved by Sema3F itself, but in the case of Fc-15F22P, the improvement of vascular permeability was induced. Also, in the case of single peptides F22 and F22P, and in the case of fusion forms with lower binding capacity, Fc-4F22 and Fc-15F22, permeability of HUVEC was not improved. - From Examples 9(1) and 9(2) above, it was confirmed that Fc-TPP improves permeability of Fc-TPP to vascular endothelial cells in vitro. Accordingly, in order to confirm the improvement of Fc-TPP's permeability in mouse models, immunohistochemistry (IHC) experiment was conducted.
- As an experiment for confirming the improvement of Fc-TPP's permeability in tumor tissues, A431 cells were injected into Balb/c nude mice with subcutaneous injection at a density of 5×106 per mouse, and when the tumor volume became about 300 to 400 mm3 after about 9 days, each of PBS, Fc, Fc-15A22P and peptide A22P in an amount of 5 mg/kg was intravenously injected. 3 hours after injection, tumors were extracted from mice to conduct immunohistochemistry experiment. The extracted tumors were cut into 20 μm thick using a frozen section method, and stained with a primary antibody, CD31 antibody (BD Pharmingen), and a TRITC (red fluorescence)-conjugated secondary antibody. Further, in order to observe Fc-TPP distributed in the tissues, FITC (green fluorescence)-conjugated antibodies recognizing Fc were used.
-
FIG. 11c illustrates a result of immunohistochemistry for confirming TPP's infiltration capacity in actual tumor tissues. As illustrated inFIG. 11c , single peptides A22 and A22P did not infiltrate into the tissues, whereas, unlike the control group Fc, the fusion forms Fc-15A22 and Fc-15A22P selectively reached tumor tissues, and they infiltrated into tumor cells. Also, it was confirmed that Fc-15A22P having higher binding capacity to neuropilin-1 and -2 than Fc-15A22 infiltrated into tissues more effectively. - Additionally, in order to confirm the improvement of vascular permeability in vivo, Evans Blue assay was conducted. As an experiment, FaDu cells were injected into Balb/c nude mice (Nara Bio, 4-week-old, female) with subcutaneous injection at a density of 5×106 per mouse, and when the tumor volume became about 500 mm3 after about 10 days, Evans Blue (Sigma) 1 mg, and each of PBS, Fc, and Fc-15A22P in an amount of 7.5 mg/kg was intravenously injected. As a control group, VEGF165A (400 ng) and
Semaphorin 3A (400 ng) were injected under same conditions. 40 minutes after injection, the mice were perfused through the heart with PBS containing 1% BSA, and tumor tissues were extracted (FIG. 11d ). Then, the extracted tissues were put into 1 ml of 2,2N-methylaformamide (Sigma) and reacted at 37° C. overnight under mild shaking condition. Thereafter, the supernatant was obtained from centrifugation to measure the absorbance (600 nm), and the amount that Evans Blue penetrated was quantified. -
FIG. 11d illustrates a result of confirming the improvement of vascular permeability through Evans Blue assay. The left panel is a photograph of cancer cells extracted, and the right panel illustrates a result of measuring absorbance (600 nm) for quantifying the Evans Blue. As illustrated inFIG. 11d , it was confirmed that VEGF165A and Sema3A increased vascular permeability as already known, and that Fc-15A22P also increased vascular permeability, unlike Fc. - In order to confirm the effect of TPP on cancer cells, a change in E-cadherin was analyzed in human head and neck cancer cell line FaDu, under the same conditions as above experiments through Western blot.
-
FIG. 11e illustrates a result of Western blot for a change in E-cadherin in human head and neck cancer cell line FaDu. As illustrated inFIG. 11e , Sema3A derived TPP did not induce a change in E-cadherin, whereas Fc-15A22 and Fc-15A22P induced a decrease in E-cadherin. Also, Sema3F induced a decrease in E-cadherin and when treated with Fc-15F22P, E-cadherin was decreased. - Upon collectively analyzing the above experimental results, Fc-TPP was constructed from peptides derived from each of Sema3A and Sema3F, but the original proteins and peptides derived therefrom did not always induce the same results in the properties of original proteins, specificity to neuropilin-1 or neuropilin-2, and tendency of signal transduction such as a decrease in VE-cadherin or E-cadherin. Further, peptides having binding capacity to neuropilin-1 and -2 did not induce signal transduction by TPP as single forms, whereas the fusion form Fc-TPP effectively induced signal transduction. This means that signal transduction by neuropilin-1 and -2 is effective in the fusion form of Fc-TPP. Also, Fc-4A/F22 did not induce signal transduction, whereas Fc-15A/F22 and Fc-15A/F22P induced signal transduction. This means that the fusion form Fc-TPP more effectively induces signal transduction by neuropilin-1 and -2, than signal form peptides, and that the effect varies depending on the length of linkers of Fc-TPP.
- (1) Tube formation assay for confirming capacity of Fc-15A22P to inhibit tube formation in HUVEC
- VEGF165A is known to form new blood vessels (angiogenesis) using neuropilin-1 as a co-receptor. As an experiment confirming angiogenesis in vitro based thereon, tube formation assay was conducted. As an experimental method, 50 μl of ECMatrix was injected into 96-well plates and polymerized at 37° C. for 2 hours. After 2 hours, HUVEC cells were suspended using Endothelial basal medium (EBM, PromoCell) and plated on ECMatrix at a density of 1×104 per well by mixing with VEGF165A (20 ng/ml), Fc, Fc-15A22P (1 μM), followed by incubation for 8 hours. The incubated cells were observed by a microscope to obtain images.
-
FIG. 12a illustrates a result confirming the capacity to inhibit tube formation through tube formation assay. As illustrated inFIG. 12a , it was confirmed that VEGF165A increased tube formation as already known, and that Fc-15A22P, unlike Fc, inhibited the tube formation. - Additionally, in order to confirm capacity to inhibit angiogenesis in vivo, matrigel plug assay was conducted. As an experimental method, 7.5×106 A431 cells per mouse, 80 μg of Fc or Fc-15A22P, and 0.4 ml of Matrigel (BD Biosciences) were subcutaneously injected into 6 to 8-week old balb/c nude mice. 9 days after injection, matrigel plug was removed to take photographs of the image (
FIG. 12b ), and cut into 20 μm thick using a frozen section method to perform immunohistochemistry experiments. Blood vessels were stained with a primary antibody, CD31 antibody, and a TRITC (red fluorescence)-conjugated secondary antibody recognizing the primary antibody, to measure the density of the blood vessels.FIG. 12b illustrates a result confirming that Fc-15A22P is capable of inhibiting angiogenesis induced by VEGF165A in mice in vivo. - (1) Vector for mAb-TPP Production, and Expression and Purification Thereof
- Example 9 above confirmed that Fc-TPP improves the permeability of vascular endothelial cells in vitro and in vivo. Accordingly, TPP was fused to Fc terminus of antibodies as a format for verifying the effect of TPP in mouse models.
-
FIG. 13(A) is a schematic diagram illustrating a fusion antibody (mAb-TPP) where a peptide binding to neuropilin is linked by a peptide linker to C-terminus of a heavy chain constant region of single clone antibody.FIG. 13(B) illustrates a schematic diagram illustrating that the fusion antibody of (A) binds to b1 domain of neuropilin membrane protein. Further,FIG. 13(C) illustrates the mechanism expected when the fusion antibody (mAb-TPP) is introduced into the body. The peptide fused antibodies bind to overexpressed neuropilin membrane proteins of tumor endothelium and various tumor cells to {circle around (1)} increase selective distribution in tumor tissues, {circle around (2)} increase extravasation, and {circle around (3)} induce signal transduction which increases infiltration into tumor tissues, thereby remarkably increasing tumor tissue specific distribution of fusion antibodies and infiltration into tumor tissues. - As a format for verifying the effect of TPP in mouse models, TPP fused Cetuximab and Trastuzumab were produced. TPP was fused to C-terminus of Fc of the existing anti-EGFR antibodies, Cetuximab IgG and Trastuzumab IgG.
FIGS. 14 and 15 are maps of exemplary vectors for expressing IgG heavy chain-TPP and IgG light chain in host cells, respectively. -
FIGS. 16(A) and 17(A) are schematic diagrams illustrating TPP fused antibodies. The expression and purification were performed in HEK293F in the same manner as in Example 4 above, and the purity was confirmed through SDS-PAGE.FIGS. 16(B) and 17(B) illustrate results of analyzing the size and purity on SDS-PAGE under reducing and non-reducing conditions, after transient expression and purification in HEK293F cells through co-transformation. - The following Table 8 shows the yield of proteins produced per 1 L culture of purified TPP fusion proteins. The results obtained from three times experiments were statistics processed, and ± represents standard deviation. The obtained yields of proteins (Cetuximab-15A22p and Trastuzumab-15A22p) are not remarkably different from those of wild-type proteins (Cetuximab and Trastuzumab).
-
TABLE 8 Clone Yield (mg/L culture) Cetuximab 39.9 ± 6.2 Cetuximab-15A22p 37.4 ± 4.7 Trastuzumab 113.6 ± 9.2 Trastuzumab-15A22p 105.8 ± 8.9
Comparison of purification yields between TPP fused Cetuximab and Trastuzumab
(2) SPR Confirming Antigen Binding Capacity of mAb-TPP - In order to confirm through surface plasmon resonance (SPR) whether mAb-TPP maintains binding affinity with antigen binding capacity of the existing antibodies, EGFR and Her2 in an amount of about 1000 RU were fixed on CM5 chips. The analysis was performed with HBS-EP buffer (10 mM Hepes, 3 mM ethylenediamine tetra acetic acid, and 0.005% surfactant P20 (pH 7.4), GE Healthcare) flowing at a rate of 30 μl/min. After bond and dissociation analysis, the regeneration of CM5 chip was performed by streaming a buffer (20 mM NaOH, 1 M NaCl, pH 10.0) at a
stream velocity 30 μl/min for 1 minute. Each sensorgram obtained by 3 minute-bond and 3 minute-dissociation was normalized and subtracted by comparing with blank cells to calculate the binding affinity. The result is shown in Table 9. As shown in Table 9, the binding affinity of Cetuximab to EGFR was analyzed almost similarly as in other references, and the binding affinity of Cetuximab-TPP to EGFR was analyzed to be similar compared with Cetuximab. Also, the binding affinity of Trastuzumab to Her2 was analyzed almost similarly as in other references, and the binding affinity of Trastuzumab-TPP to Her2 was analyzed to be similar compared with Trastuzumab. It was confirmed that when TPP was fused to mAb, the fusion did not affect the existing antigen binding of the antibody. The analysis was performed using at least five sensorgrams, and the results obtained from two times experiments were statistics processed, and ± represents standard deviation of independent experimental results. -
TABLE 9 Association Dissociation Binding Recep- rate rate affinity Clone tors ka (M−1s−1) kd (S−1) KD(nM) Cetuximab EGFR 6.24 ± 0.17 × 106 1.50 ± 0.35 × 10−3 0.24 ± 0.02 Cetuximab- EGFR 7.17 ± 0.12 × 106 1.53 ± 0.37 × 10−3 0.21 ± 0.01 15A22p Trastuzumab Her2 7.90 ± 0.07 × 104 1.06 ± 0.14 × 10−5 0.14 ± 0.01 Trastuzumab- Her2 8.45 ± 0.31 × 104 1.67 ± 0.37 × 10−5 0.20 ± 0.05 15A22p
Analysis result of binding affinity of Cetuximab and Trastuzumab to EGFR and Her2
(3) Sandwich ELISA for Confirming Bispecificity of mAb-TPP - Sandwich ELISA was performed to confirm bispecificity of mAb-TPP.
- Specifically, 1 μg of each of b1b2 domains (273-586) of neuropilin-1 and b1b2 domain (275-595) of neuropilin-2 were bound at room temperature for 1 hour in 96-well EIA/RIA plates (COSTAR Corning In., USA), and then washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA). After binding for 1 hour with 5% skim milk (pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA), it was washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA). The mAb and mAb-TPP at a concentration of 100 nM were bound to b1b2 domains of neuropilin-1 and -2, and then washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA). The bionylated EGFR (SIGMA-ALDRICH co., USA) and Her2-ECD (R&D systems, Minneapolis, Minn.) were diluted to be 1 μM to 1 nM and bound in each well using the same manner as above, and then washed three times for 10 minutes with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, SIGMA-ALDRICH co., USA). After binding to alkaline phosphatase (AP)-conjugated anti-human mAb (Sigma, USA) and reacting with pnitrophenyl palmitate (pNPP, SIGMA-ALDRICH co., USA), absorbance at 405 nm was quantified.
-
FIGS. 16(C) and 17(C) are results confirming from ELISA analysis the bispecificity of mAb-TPP which has binding capacity to neuropilin-1 or -2 and original antigens of mAb simultaneously. - (4) SPR for Confirming Binding Capacity of mAb-TPP to Fc Receptor
- In order to confirm through surface plasmon resonance (SPR) whether mAb-TPP maintains binding capacity to Fc receptors of the existing antibodies, the affinity with FcγRIIa, FcγRIIIa, FcγRIIIb, and FcRn was analyzed. For FcγRIIa, FcγRIIIa, and FcγRIIIb, the same method as in Example 6 above was used. For FcRn, the wild-type antibodies have the properties of binding to FcRn only at pH 6.0, but not at pH 7.4. For affinity analysis, SPR was performed for FcRn of mAb and mAb-TPP under pH 6.0 condition. FcRn in an amount of about 1000 RU was fixed on CM5 chips. The analysis was performed with PBST buffer (pH 6.0, 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, 0.005% surfactant P20, SIGMA-ALDRICH co., USA) flowing at a rate of 30 μl/min. After bond and dissociation analysis, the regeneration of CM5 chip was performed by streaming HBS-EP buffer (10 mM Hepes, 3 mM ethylenediamine tetra acetic acid, and 0.005% surfactant P20 (pH 8.0), GE Healthcare) at a
stream velocity 30 μl/min for 1 minute. Each sensorgram obtained by 3 minute-bond and 3 minute-dissociation of mAb (Cetuximab, Trastuzumab) and mAb-TPP (Cetuximab-TPP, Trastuzumab-TPP) was normalized and subtracted by comparing with blank cells to calculate the binding affinity. The result is shown in Table 10. - The difference was confirmed in binding between this sensorgram and the sensorgram at the highest concentration 625 nM among the concentrations analyzed at pH 6.0 of the sensorgrams analyzed in Example 6 above.
FIG. 18 illustrates a result confirming whether the binding capacities of mAb and mAb-TPP to FcRn are similar to each other depending on pH. As illustrated inFIG. 18 , it was confirmed that all of mAb (Cetuximab, Trastuzumab) and mAb-TPP (Cetuximab-TPP, Trastuzumab-TPP) have the properties of binding to FcRn only at pH 6.0, but not at pH 7.4. Accordingly, it was confirmed that TPP fusion did not affect the binding capacity to Fc receptor. -
TABLE 10 Association rate Dissociation rate Binding affinity Clone Receptor ka (M−1s−1) kd (S−1) KD(M) Cetuximab FcRn (pH 6.0) 8.27 ± 0.60 × 103 4.28 ± 0.16 × 10−3 5.17 ± 0.21 × 10−7 Cetuximab-15A22p FcRn (pH 6.0) 7.71 ± 0.04 × 103 4.35 ± 0.06 × 10−4 5.64 ± 0.10 × 10−7 Trastuzumab FcRn (pH 6.0) 3.24 ± 0.07 × 103 1.49 ± 0.14 × 10−3 4.59 ± 0.16 × 10−7 Trastuzumab-15A22p FcRn (pH 6.0) 7.23 ± 0.30 × 103 3.72 ± 0.31 × 10−3 5.15 ± 0.15 × 10−7 Cetuximab FcγR IIa 8.64 ± 0.04 × 103 2.28 ± 0.17 × 10−3 2.64 ± 0.14 × 10−7 Cetuximab-15A22p FcγR IIa 1.40 ± 0.07 × 104 3.28 ± 0.14 × 10−3 3.12 ± 0.16 × 10−7 Trastuzumab FcγR IIa 2.39 ± 0.20 × 104 5.04 ± 0.25 × 10−3 2.11 ± 0.10 × 10−7 Trastuzumab-15A22p FcγR IIa 2.47 ± 0.17 × 104 7.98 ± 0.35 × 10−3 3.23 ± 0.27 × 10−7 Cetuximab FcγR IIIa 1.94 ± 0.19 × 104 1.91 ± 0.20 × 10−2 9.85 ± 0.12 × 10−7 Cetuximab-15A22p FcγR IIIa 1.98 ± 0.20 × 104 1.68 ± 0.25 × 10−2 8.47 ± 0.21 × 10−7 Trastuzumab FcγR IIIa 1.61 ± 0.28 × 104 1.50 ± 0.20 × 10−2 9.33 ± 0.85 × 10−7 Trastuzumab-15A22p FcγR IIIa 2.23 ± 0.28 × 104 2.09 ± 0.03 × 10−2 9.38 ± 0.17 × 10−7 Cetuximab FcγR IIIb 5.10 ± 0.45 × 103 4.20 ± 0.19 × 10−2 8.23 ± 0.14 × 10−6 Cetuximab-15A22p FcγR IIIb 1.57 ± 0.45 × 103 1.45 ± 0.20 × 10−2 9.22 ± 0.22 × 10−6 Trastuzumab FcγR IIIb 5.30 ± 0.14 × 103 4.47 ± 0.22 × 10−2 8.43 ± 0.33 × 10−6 Trastuzumab-15A22p FcγR IIIb 7.08 ± 0.43 × 103 6.66 ± 0.13 × 10−2 9.39 ± 0.23 × 10−6 Analysis of binding affinity KD values of IgG-TPP to Fc receptor - In order to confirm the infiltration of antibodies within tumor tissues by IgG-TPP constructed in the above experiments, 5×106 cells of each of FaDu and A431 were subcutaneously injected into Balb/c nude mice respectively, and when the volume of the tumor became about 300-400 mm3 after about 9 days, 2.5 mg/kg of PBS, Cetuximab, and Cetuximab-15A22P were intravenously injected, respectively. After the injection, the tumor was extracted from the mice after 3 hours and 12 hours, respectively, and an immunohistochemistry experiment was performed. The tissue was stained and observed in the same manner as in Example 9.
-
FIG. 19a illustrates a result of IHC for confirming the infiltration capacity of Cetuximab-TPP in tumor tissues. As illustrated inFIG. 19a , in the case of Cetuximab, green fluorescence was observed only around the blood vessels in the two cancer cell tissues of FaDu and A431. In comparison, it was confirmed that in the case of Cetuximab-15A22P, as compared to Cetuximab, Cetuximab-15A22P infiltrated into the tissue to be further away from the blood vessels. Also, the samples were compared after 3 hours and 12 hours, and accordingly it was confirmed that the cell infiltrates further into the tissue as time passes. ImageJ was used to quantify this. - Further, Western blot was performed from the cancer cell tissue used in this condition, and the result is illustrated in
FIG. 19b . As illustrated inFIG. 19b , when performing Western blot using antibodies recognizing the heavy chain site and light chain site of human antibody, respectively, it was confirmed that more amount of Cetuximab-15A22P was present in the tissue than Cetuximab. Also, it was confirmed that only E-cadherin was decreased by Cetuximab-TPP, and N-cadherin, vimentin, fibronetin, etc., which are proteins relating to metastasis of cancer cells, did not change. Unlike the metastasis of cancer cells accompanied by the decrease of E-cadherin and increase of N-cadherin, vimentin, fibronetin, etc., it was indirectly proved that metastasis and infiltration of cancer cells do not occur by the effect of TPP. - Also, in order to confirm whether such tendency actually affects inhibition of cancer cells, an experiment confirming the inhibition of cancer cell growth was performed in nude mice. In the same manner as Example 9, cell line FaDu was subcutaneously injected into nude mice, and after about 5 days from transplanting cells, when the volume of the tumor became about 120 mm3, Cetuximab and Cetuximab-15A22P were intravenously injected 6 times every 3 days in an amount of 2.5 and 10 mg/kg (N=6).
- As illustrated in
FIG. 19c , as compared to the control group PBS, it was confirmed that Cetuximab (CTX) and Cetuximab-15A22P inhibited cancer cell growth, and that Cetuximab-15A22P inhibited it more effectively than Cetuximab. Also, as illustrated inFIG. 19d , it was confirmed that the experimental group Cetuximab-15A22P did not present a great difference in weight as compared with the experimental group Cetuximab, and accordingly, it is determined to have no toxicity. - In the same manner as the above test, an IHC experiment was performed to confirm the antibody infiltrated into tumor tissue using the constructed Trastuzumab-15A22P. 5×106 cells of SK-OV-3 were subcutaneously injected into Balb/c nude mice, respectively, and when the volume of the tumor became about 300-400 mm3 after about 9 days, 2.5 mg/kg of PBS, Trastuzumab and Trastuzumab-15A22P were intravenously injected, respectively. After the injection, the tumor was extracted from the mice after 3 hours and 12 hours, respectively, and an immunohistochemistry experiment was performed. The tissue was stained and observed in the same manner as the experiment above.
-
FIG. 20a illustrates a result of IHC for confirming the infiltration capacity of Trastuzumab-TPP in tumor tissues. As illustrated inFIG. 20a , in the case of Trastuzumab, green fluorescence was observed only around the blood vessels. In comparison, in the case of Trastuzumab-15A22P, as compared to Trastuzumab, it is confirmed that Trastuzumab-15A22P infiltrated into the tissue to be further away from the blood vessels. Also, the samples were compared after 3 hours and 12 hours, and accordingly it was confirmed that the cell infiltrates further into the tissue as time passes. ImageJ was used to quantify this. - Further, Western blot was performed from the cancer cell tissue used in this condition, and the result is illustrated in
FIG. 20b . As illustrated inFIG. 20b , when performing Western blot using antibodies recognizing the heavy chain site and light chain site of human antibody, respectively, it was confirmed that more amount of Trastuzumab-15A22P was present in the tissue than Trastuzumab. Also, it was confirmed that only E-cadherin was decreased by Trastuzumab-TPP, and N-cadherin, vimentin, fibronetin, etc., which are proteins relating to metastasis of cancer cells, did not change. Through this, it was indirectly proved that metastasis and infiltration of cancer cells do not occur by the effect of TPP. - In the same manner as the above experiment, in order to confirm the effect of cancer cell growth inhibition of Trastuzumab-TPP, 5×106 cells of SK-OV-3 were subcutaneously injected into nude mice, respectively, and when the volume of the tumor became about 80 mm3 after about 7 days from transplanting the cells, Trastuzumab and Trastuzumab-15A22P were intravenously injected 6 times every 3 days in an amount of 2.5 and 5 mg/kg (N=6).
FIG. 20c illustrates a result of measuring the volume of the tumor to confirm the effect of Trastuzumab-TPP to inhibit cancer cell growth. As illustrated inFIG. 20c , it was confirmed that Trastuzumab-15A22P induced inhibition of cancer cell growth more effectively than Trastuzumab.FIG. 20d illustrates a result of measuring the weight of mice during the experiment. As illustrated inFIG. 20d , it can be understood that the experimental group Trastuzumab-15A22P did not present a great difference in weight as compared with the experimental group Trastuzumab, and it is determined to have no toxicity. - The results show that tumor tissue-penetrating A22p peptide may be generally applied to various monoclonal antibodies recognizing various antigens.
- As a result, it is confirmed that improvement of infiltration capacity of TPP in tissue inhibits the growth of cancer cells in mice. In order to confirm whether the TPP itself has cytotoxicity in vitro, the cell lines used in the experiments above, FaDu and SK-OV-3, are treated with Fc-TPP and mAb-TPP, respectively, and the degree of cell growth inhibition was evaluated.
- Particularly, 1×104 cells (FaDu, SK-OV-3) were diluted in 0.5 ml of a medium including 10% FBS per well in 24-well plate, respectively, and cultured. When the cells were stabilized, they were treated with 1 μM of Fc, Fc-15A22P, mAb, and mAb-15A22P, and observed for 72 hours, and then the number of cells alive was counted to compare the degree of cell growth. The effect was confirmed by treating cell line FaDu with Fc, Fc-15A22P, Cetuximab, and Cetuximab-15A22P, and treating SK-OV-3 with Fc, Fc-15A22P, Trastuzumab, and Trastuzumab-15A22P.
-
FIG. 21 illustrates a result of in vitro evaluation on the degree of cell growth inhibition by treating FaDu and SK-OV-3 with Fc-TPP and mAb-TPP. As illustrated inFIG. 21 , Cetuximab and Cetuximab-15A22P inhibited cell growth by about 30% in cell line FaDu, and Trastuzumab and Trastuzumab-15A22P inhibited cell growth by about 30-35% in cell line SK-OV-3. It was confirmed that there was no difference between the mAb and mAb-TPP. Likewise, Fc and Fc-TPP did not inhibit cell growth in both FaDu and SK-OV-3. This means that with regard to the effect of TPP by neuropilin, TPP maximizes the effect by increasing infiltration into tumor tissue without directly affecting the growth of cancer cells. Also, it was indirectly confirmed that the effect of mAb-TPP confirmed through the above test is not an effect by the interaction between EGFR and neuropilin, and between HER2 and neuropilin. - An aspect of the present invention provides a tumor tissue-penetrating peptide (TPP) specifically binding to neuropilin.
- The tumor tissue-penetrating property in the above aspect means, for example, having any one of the properties of 1) specifically recognizing a tumor specific vascular endothelial cell, a tumor cell or tissue and accumulating in it, 2) widening intercellular gaps between tumor vascular endothelial cells and promoting extravasation, and 3) controlling intercellular gap between tumors within the tumor and increasing infiltration within the tumor.
- The term neuropilin (NRP) as used herein is a transmembrane glycoprotein, and there are two forms of neuropilin, NRP1 and NRP2. The structures of NRP1 and NRP2 are as illustrated in
FIGS. 1 (D) and (E). Neuropilin broadly consists of five domains, and from the N-terminus, a1 and a2 domains are classified as CUB domains, and an Ig-like C2 type of semaphorin binds thereto. Particularly, this site forms a complex with plexin, and plays a role of increasing the binding force with semaphorin-plexin. The b1 and b2 domains of neuropilin are classified as FV/VIII domains, and the C-terminus of VEGF family ligand or secretedclass 3 semaphorin ligands (secreted Sema3s) binds thereto. The VEGF ligand and Sema3s have a site recognizing furin hydrolysis enzymes (RXRR, Arg-X-Arg-Arg), and thus they commonly end with an arginine (Arg) amino acid residue at the C-terminus by Furin processing (Adams et al. 1997). It has been reported that the C-terminus Arg residues of VEGF ligand and Sema3s are very important in the interaction between b1 and b2 domains of neuropilin (Teesalu et al. 2009). The tertiary structure of the complex of VEGF ligand and b1b2 domains of neuropilin has been revealed (Parker et al., 2012), and accordingly the amino acid sequence of VEGF important in the binding to the b1b2 domains of neuropilin may be known. However, for Sema3A binding to NRP1 and Sema3F binding to NRP2, it still has not been found out which site of the C-terminus of these ligands specifically bind to NRP. - The tumor tissue-penetrating peptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4 is a peptide designed based on the similarity of the C-terminus sequences by analyzing the sequence of the VEGF165A and the furin C-terminus sequence of
semaphorin 3A andsemaphorin 3F known to bind to neuropilin, by analyzing the amino acid sequence and length of the binding site of VEGF165A ligand binding to b1b2 domains of neuropilin. - A22, which is a tumor tissue-penetrating peptide represented by an amino acid sequence of SEQ ID NO:1, is a peptide consisting of 22 amino acids derived from residues 739-760, which are part of the basic domain of
semaphorin 3A. A22p, which is a tumor tissue-penetrating peptide represented by an amino acid sequence of SEQ ID NO: 2, transforms the third amino acid from the C-terminus of A22, which is the 758th asparagine (Asn758), to proline (Pro, P) (Asn758Pro). F22, which is a tumor tissue-penetrating peptide represented by an amino acid sequence of SEQ ID NO: 3, is a peptide consisting of 22 amino acids derived from residues 758-779, which are part of the basic domain ofsemaphorin 3F. F22p, which is a tumor tissue-penetrating peptide represented by an amino acid sequence of SEQ ID NO: 4, transforms the third amino acid from the C-terminus of F22, which is the 777th asparagine (Asn777), to proline (Pro, P) (Asn777Pro). The peptides A22p and F22p are designed to derive peptides with improved affinity with neuropilin by inserting mutants to Sema3A- and Sema3F-derived peptides. - The name of the peptide of the present invention, semaphorin-derived sequence and SEQ ID NO. are as shown below.
-
Entire amino acid sequence from TPP Ligand N-terminus SEQ ID name derived to C-terminus) NO. A22 Semaphor HTPGNSNKWKH SEQ ID in 3A LQENKKGRNRR NO: 1 A22p Semaphor HTPGNSNKWKH SEQ ID in 3A LQENKKGRPRR NO: 2 F22 Smephor REAPGAPRSPE SEQ ID in 3F PQDQKKPRNRR NO: 3 F22p Semaphor REAPGAPRSPE SEQ ID in 3F PQDQKKPRPRR NO: 4 - The tumor tissue-penetrating peptide of the above aspect may further include a linker peptide. The linker peptide may consist of 1 to 50 amino acids, preferably 4 to 20 amino acids, and more preferably 4 to 15 amino acids. Also, the linker peptide may consist of glycine, serine or alanine, preferably the sequence of the linker peptide may consist of an amino acid sequence of (GA)n or (GGGGS)m (wherein n and m are each independently an integer between 1 and 20), and more preferably may consist of an amino acid sequence of GAGA or (GGGGS)3.
- The tumor targeting capacity is confirmed in various combinations of the linker and semaphorin-derived sequence in a detailed embodiment of the present invention. The name of peptide, sequence of linker, semaphorin-derived sequence, length of entire amino acid and SEQ ID NO. are as shown below.
-
Entire amino acid sequence Length (from N-terminus of to C-terminus) entire TPP Linker Neuropilin amino SEQ ID Name sequence target sequence acid NO. 4A22 GAGA HTPGNSNKWKHLQEN 26 SEQ ID KKGRNRR (SEQ NO: 5 ID NO: 1) 15A22 GGGGSGGG HTPGNSNKWKHLQEN 37 SEQ ID GSGGGGS KKGRNRR (SEQ NO: 6 ID NO: 1) 15A22p GGGGSGGG HTPGNSNKWKHLQEN 37 SEQ ID GSGGGGS KKGRPRR (SEQ NO: 7 ID NO: 2) 4F22 GAGA REAPGAPRSPEPQDQ 26 SEQ ID KKPRNRR (SEQ NO: 8 ID NO: 3) 13F22 GGGGSGGG REAPGAPRSPEPQDQ 37 SEQ ID GSGGGGS KKPRNRR (SEQ NO: 9 ID NO: 3) 15F22p GGGGSGGG REAPGAPRSPEPQDQ 37 SEQ ID GSGGGGS KKPRPRR (SEQ NO: 10 ID NO: 4) - Another aspect of the present invention provides a fusion protein, a nanoparticle or a liposome having the tumor tissue-penetrating peptide fused therein.
- The protein may be antibodies, antibody fragments, immuoglubulin, peptides, enzymes, transcription factors, toxins, antigen peptides, hormones, carrier proteins, structural proteins, motor function proteins, receptors, signaling proteins, storage proteins, membrane proteins, transmembrane proteins, internal proteins, external proteins, secretory proteins, viral proteins, native proteins, glycoproteins, cleaved proteins, proteins with disulfide bond, protein complexes, chemically modified proteins, or prions, etc.
- According to the present invention, liposomes include at least one lipid bilayer enclosing the inner aqueous compartment, which is capable of being associated by itself. Liposomes may be characterized by membrane type and size thereof. Small unilamellar vesicles (SUVs) may have a single membrane and may range between 20 and 50 nm in diameter. Large unilamellar vesicles (LUVs) may be at least 50 nm in diameter. Oliglamellar large vesicles and multilamellar large vesicles may have multiple, usually concentric, membrane layers and may be at least 100 nm in diameter. Liposomes with several nonconcentric membranes, i.e., several small vesicles contained within a larger vesicle, are referred to as multivesicular vesicles.
- According to the present invention, a nanoparticle refers to a particle including substances ranging between 1 and 1,000 nm in diameter. The nanoparticle may be a metal nanoparticle, a metal/metal core shell complex consisting of a metal nanoparticle core and a metal shell enclosing the core, a metal/non-metal core shell consisting of a metal nanoparticle core and a non-metal shell enclosing the core, or a non-metal/metal core shell complex consisting of a non-metal nanoparticle core and a metal shell enclosing the core. According to an embodiment, the metal may be selected from gold, silver, copper, aluminium, nickel, palladium, platinum, magnetic iron and oxides thereof, but is not limited thereto, and the non-metal may be selected from silica, polystyrene, latex and acrylate type substances, but is not limited thereto.
- Also, the tumor tissue-penetrating peptide may have at least a bivalent binding to neuropilin.
- According to the present invention, fusion refers to unifying two molecules having the same or different function or structure, and the methods of fusing include any physical, chemical or biological method binding the tumor tissue-penetrating peptide to the protein, nanoparticle or liposome. Preferably, the fusion may be made by a linker peptide, and for example, the linker peptide may bind to C-terminus of Fc fragment of an antibody.
- In the present invention, a complete antibody has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to each heavy chain by a disulfide bond (SS-bond). A constant region of the antibody is divided into a heavy-chain constant region and a light-chain constant region, and the heavy-chain constant region has γ, μ, α, δ and ε types, and γ1, γ2, γ3, γ4, α1 and α2 subclasses. The light-chain constant region has κ and λ types.
- The term “heavy chain” as used herein may be interpreted to include a full-length heavy chain including variable region domain VH including an amino acid sequence having a variable region sequence sufficient to confer antigen-specificity and three constant region domains CH1, CH2 and CH3, and a fragment thereof. Also, the term “light chain” as used herein may be interpreted to include a full-length light chain including a variable region domain VL including an amino acid sequence having a variable region sequence sufficient to confer antigen-specificity and constant region domain CL, and a fragment thereof.
- According to the present invention, a fragment of an antibody refers to each domain of a heavy chain or a light chain of an antibody, or a fragment thereof. For example, it may be a heavy-chain constant region, a heavy-chain variable region, a light-chain constant region, or a light-chain variable region of an antibody, or a fragment thereof. Preferably, the fragment of the antibody may be a heavy-chain constant region of an antibody.
- Also, the fragment of the antibody may be a monomer, a dimer or a polymer.
- The antibody includes monoclonal antibodies, non-specific antibodies, non-human antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFV) and anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of these antibodies, but is not limited thereto.
- The monoclonal antibody may be IgG, IgM, IgA, IgD or IgE. For example, the monoclonal antibody may be IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type, and may be IgG1. Also, the light-chain constant region of the antibody may be λ or κ type.
- The peptide may bind to a heavy-chain constant region (Fc) fragment of an antibody, preferably to the C-terminus of a heavy-chain constant region (Fc) fragment of an antibody, and the binding may be formed by a linker peptide.
- Also, another aspect of the present invention provides a polynucleotide coding a peptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 10.
- The term “polynucleotide” as used herein refers to a deoxyribonucleotide or ribonucleotide polymer present in single-stranded or double-stranded form. It includes RNA genome sequence, DNA (gDNA and cDNA), and RNA sequence transcribed therefrom. Unless otherwise described, it also includes an analog of the natural polynucleotide.
- The polynucleotide includes not only a nucleotide sequence coding an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 10, but also a complementary sequence thereto. The complementary sequence includes a sequence fully complementary and a sequence substantially complementary. For example, this means a sequence that may be hybridized with a nucleotide sequence coding an amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 10 under stringent conditions known in the pertinent art.
- Also, the polynucleotide may be modified. The modification includes the addition, deletion, or non-conservative substitution or conservative substitution of nucleotides. The polynucleotide coding the amino acid sequence is interpreted to include a nucleotide sequence that has substantial identity to the nucleotide sequence. The substantial identity may refer to a sequence having at least 80% sequence identity, at least 90% sequence identity, or at least 95% sequence identity when aligning the nucleotide sequence to correspond to any other sequence as much as possible and analyzing the aligned sequence using an algorithm generally used in the pertinent art.
- Another aspect of the present invention provides a recombinant vector including the polynucleotide.
- The term “vector” as used herein refers to a means for expressing a target gene in a host cell. For example, the vector may include plasmid vector, cosmid vector, bacteriophage vector, and virus vectors such as adenovirus vector, retrovirus vector, and adeno-associated virus vector. The vector that may be used as the recombinant vector may be produced by operating plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), phages (for example, λgt4λB, λ-Charon, λΔz1 and M13, etc.), or virus (for example, CMV, SV40, etc.) commonly used in the pertinent art.
- A polynucleotide coding an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 10 in the recombinant vector may be operatively linked to a promoter. The term “operatively linked” as used herein means a functional linkage between a nucleotide expression control sequence (such as a promoter sequence) and a second nucleotide sequence. Accordingly, the control sequence may control the transcription and/or translation of the second nucleotide sequence.
- The recombinant vector may be generally constructed as a vector for cloning or a vector for expression. As the vector for expression, vectors generally used for expressing foreign protein from plants, animals or microorganisms in the pertinent art may be used. The recombinant vector may be constructed by various methods known in the pertinent art.
- The recombinant vector may be constructed to be a vector that employs a prokaryotic cell or an eukaryotic cell as a host. For example, when the vector used is an expression vector and employs a prokaryotic cell as a host, the vector generally includes a strong promoter which may promote transcription (for example, pLλ promoter, tip promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and termination sequences for transcription/translation. When the vector employs an eukaryotic cell as a host, a replication origin operating in the eukaryotic cell included in the vector may include an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, a CMV replication origin and a BBV replication origin, etc., but is not limited thereto. In addition, a promoter derived from a genome of a mammal cell (for example, a metalthionine promoter) or a promoter derived from a virus of a mammal cell (for example, an adenovirus anaphase promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a cytomegalo virus (CMV) promoter, or a tk promoter of HSV) may be used, and the promoter generally has a polyadenylated sequence as a transcription termination sequence.
- Meanwhile, in addition to the tumor tissue-penetrating peptide of the present invention, the vector may express an antibody having the peptide fused therein or a fragment thereof. In the case of an antibody having the peptide fused therein or a fragment thereof, the vector may use both a vector system expressing a peptide and an antibody or fragment thereof simultaneously in one vector, or a vector system expressing them in separate vectors. For the latter, the two vectors may be introduced into the host cell through co-transformation and targeted transformation.
- For example, the recombinant vector of the present invention may have the cleavage map illustrated in
FIG. 4(B) orFIG. 13(B) . - Another aspect of the present invention provides a host cell transformed with the recombinant vector.
- Any kind of host cell known in the pertinent art may be used as a host cell. Examples of a prokaryotic cell include strains belonging to the genus Bascillus such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bascillus subtilus and Bascillus thuringiensis, Salmonella typhimurium, intestinal flora and strains such as Serratia marcescens and various Pseudomonas Spp., etc. In addition, when the vector is transformed in an eukaryotic cell, a host cell such as Saccharomyce cerevisiae, an insect cell, a plant cell, and an animal cell, for example, SP2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RN, and MDCK cell line, etc., may be used.
- Another aspect of the present invention provides a method for preparing a tumor tissue-penetrating peptide, including culturing the host cell.
- The polynucleotide or a recombinant vector including the same may be inserted into a host cell using an insertion method well known in the pertinent art. For example, when a host cell is a prokaryotic cell, the transfer may be carried out according to CaCl2 method or an electroporation method, etc., and when a host cell is an eukaryotic cell, the vector may be transferred into a host cell according to a microscope injection method, calcium phosphate precipitation method, an electroporation method, a liposome-mediated transformation method, and a gene bombardment method, etc., but the transferring method is not limited thereto. When using microorganisms such as E. coli, etc. the product ability is higher than using animal cells. However, although it is not suitable for production of intact Ig form of antibodies due to glycosylation, it may be used for production of antigen binding fragments such as Fab and Fv.
- The method for selecting the transformed host cell may be readily carried out according to a method well known in the pertinent art using a phenotype expressed by a selected label. For example, when the selected label is a specific antibiotic resistance gene, the transformant may be readily selected by culturing the transformant in a medium containing the antibiotic.
- The aspect is a concept including the preparation of a tumor tissue-penetrating peptide (TPP), and an antibody having the peptide fused therein or a fragment thereof.
- An example of a method for preparing a heavy-chain constant region (Fc) fragment of an antibody having a tumor tissue-penetrating peptide (TPP) fused therein is as shown below:
- 1) having the TPP fused in a heavy-chain constant region (Fc) fragment of an antibody starting from a hinge of an antibody and preparing a recombinant TPP fused heavy-chain constant region (Fc-TPP) expression vector cloning nucleic acids of (hinge)-CH2-CH3-linker-TPP;
- 2) transforming the prepared expression vector in a cell and expressing recombinant Fc-TPP protein; and
- 3) purifying and collecting the expressed recombinant Fc-TPP protein.
- Also, an example of a method for preparing an antibody having a tumor tissue-penetrating peptide fused therein is as shown below:
- 1) preparing a heavy-chain site of recombinant TPP fused IgG cloning nucleic acids of VH-CH1-hinge-CH2-CH3-linker-TPP and a light-chain site vector of an antibody cloning nucleic acids of VL-CL of the prepared IgG-TPP;
- 2) co-transforming the prepared heavy-chain and light-chain expression vectors in a cell, and expressing recombinant IgG-TPP protein; and
- 3) purifying and collecting the expressed recombinant IgG-TPP protein.
- Also, an aspect of the present invention provides a composition for treating or preventing cancer or angiogenesis-related diseases, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- The tumor tissue-penetrating peptide of the present invention specifically binds to neuropilin, thereby being specifically distributed in tumors, and exhibiting efficacy to penetrate into tumors. Also, a specific part is substituted with a sequence of vascular endothelial growth factor A (VEGF165A), thereby maintaining tumor infiltration effect and remarkably improving affinity to neuropilin. Also, the tumor tissue-penetrating peptide of the present invention allows the removal of heparin binding site, thereby minimizing nonspecific binding.
- The antibody having the tumor tissue-penetrating peptide fused therein of the present invention shows a production yield similar to wild-type antibody, and has the property of a bispecific antibody that can target two types of antigens, an antigen to which the antibody binds and neuropilin to which the tumor tissue-penetrating peptide binds. Accordingly, it may allow an antibody to reach a tumor tissue with high efficiency, and thus is expected to have a high effect in treating cancer.
- Also, an antibody having the tumor tissue-penetrating peptide fused therein or a fragment thereof maintains the antigen binding capacity which the wild-type antibody originally has, unique function of heavy-chain constant region (Fc), i.e., binding with FcγRn (neonatal Fc receptor) and FcRs (Fc gamma receptors), and accordingly has a long serum half-life. Also, it has an advantage that the binding site (protein A and protein G) during the purification process is preserved, and the antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity may be maintained.
- Also, the tumor tissue-penetrating peptide specifically binds to neuropilin, and competes with VEGF165A binding to neuropilin. Accordingly, it inhibits the angiogenesis function caused by VEGF165A binding to neuropilin, and thus is expected to have an effect of treating both cancer and angiogenesis-related diseases.
- The cancer may be selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of lung, squamous cell carcinoma of lung, peritoneal cancer, skin cancer, skin or ocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, pancreatic cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, liver cancer and head and neck cancer.
- The angiogenesis-related disease may be selected from the group consisting of diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginalkeratolysis, trauma, rheumatoid arthritis, systemic lupus, polyarteritis, Wegeners sarcoidosis, scleritis, Steven's Johnson disease, periphigoid radial keratotomy, corneal graft rejection, keloid, wound granulation, and glomerulonephritis.
- When the composition is prepared as a pharmaceutical composition for preventing or treating cancer or angiogenesis-related diseases, the composition may include a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier included in the composition may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, minute crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil, etc., but are not limited thereto. In addition to the above ingredients, the pharmaceutical composition may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspension, a preservative, etc.
- The pharmaceutical composition for preventing or treating cancer or angiogenesis-related diseases may be administered orally or parenterally. Such a parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, nasal administration, intrapulmonary administration, intrarectal administration, etc. Because a protein or peptide is digested when administered orally, it is preferred that a composition for oral administration is formulated to coat an active substance or to be protected against degradation in stomach. Also, the pharmaceutical composition may be administered by any device which can transport active substances to target cells.
- Proper dose of the pharmaceutical composition for preventing or treating cancer or angiogenesis-related diseases may vary according to various factors such as method for formulating, administration method, age, weight, gender, pathological state of patient, food, administration time, administration route, excretion rate and reaction sensitivity, etc. Preferably, a proper dose of the composition is within the range of 0.001 and 100 mg/kg based on an adult. The term “pharmaceutically effective dose” as used herein refers to an amount sufficient to prevent or treat cancer or angiogenesis-related diseases.
- The composition may be formulated with pharmaceutically acceptable carriers and/or excipients according to a method that can be easily carried out by those skilled in the art, and may be provided in a unit-dose form or enclosed in a multiple-dose vial. Here, the formulation may be in the form of a solution, a suspension, syrup or an emulsion in oily or aqueous medium, or may be extracts, powders, granules, tablets or capsules, and may further include a dispersion agent or a stabilizer. Also, the composition may be administered individually or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Meanwhile, the composition includes an antibody or an antigen-binding fragment, and thus may be formulated into immuno liposome. Liposome including an antibody may be prepared according to a method well known in the pertinent art. The immuno liposome is a lipid composition including phosphatidylcholine, cholesterol and polyethyleneglycol-derived phosphatidylethanolamine, and may be prepared by reverse phase evaporation method. For example, a Fab′ fragment of antibody may be conjugated to liposome through disulphide exchange reaction. Liposome may further include chemical therapeutic agents such as Doxorubicin.
- Also, an aspect of the present invention provides a composition for diagnosing cancer, including the tumor tissue-penetrating peptide, or a fusion protein, a small molecule drug, a nanoparticle, or a liposome having the peptide fused therein.
- The term “diagnosing” as used herein refers to demonstrating the presence or characteristic of a pathophysiological condition. Diagnosing in the present invention refers to demonstrating the onset and progress of cancer.
- The tumor tissue-penetrating peptide may bind to a fluorescent substance for molecular imaging in order to diagnose cancer through images.
- The fluorescent substance for molecular imaging refers to all substances generating fluorescence. Preferably, red or near-infrared fluorescence is emitted, and more preferably, a fluorescence with high quantum yield is emitted. However, the fluorescence is not limited thereto.
- Preferably, the fluorescent substance for molecular imaging is a fluorescent substance, a fluorescent protein or other substances for imaging, which may bind to the tumor tissue-penetrating peptide, but is not limited thereto.
- Preferably, the fluorescent substance is fluorescein, BODYPY, tetramethylrhodamine, Alexa, cyanine, allopicocyanine, or a derivative thereof, but is not limited thereto.
- Preferably, the fluorescent protein is Dronpa protein, enhanced green fluorescence protein (EGFP), red fluorescent protein (DsRFP), Cy5.5, which is a cyanine fluorescent substance presenting near-infrared fluorescence, or other fluorescent proteins, but is not limited thereto.
- Preferably, other substances for imaging are ferric oxide, radioactive isotope, etc., but are not limited thereto, and they may be applied to imaging equipment such as MR, PET.
Claims (33)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2013-0058619 | 2013-05-23 | ||
KR20130058619 | 2013-05-23 | ||
PCT/KR2014/004571 WO2014189303A1 (en) | 2013-05-23 | 2014-05-22 | Trans-tumoral peptide specific to neuropilin and fusion protein having same peptide fused therein |
Publications (2)
Publication Number | Publication Date |
---|---|
US20160130315A1 true US20160130315A1 (en) | 2016-05-12 |
US9975933B2 US9975933B2 (en) | 2018-05-22 |
Family
ID=51933799
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/893,317 Active 2034-09-14 US9975933B2 (en) | 2013-05-23 | 2014-05-22 | Tumor tissue-penetrating peptide specific to neuropilin and fusion protein having same peptide fused therein |
Country Status (5)
Country | Link |
---|---|
US (1) | US9975933B2 (en) |
EP (1) | EP3000825A4 (en) |
JP (2) | JP6391676B2 (en) |
KR (1) | KR101551299B1 (en) |
WO (1) | WO2014189303A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10364289B2 (en) | 2014-12-03 | 2019-07-30 | Samsung Life Public Welfare Foundation | Antibody binding to neuropilin 1 and use thereof |
WO2021068962A1 (en) * | 2019-10-11 | 2021-04-15 | 田中纯美 | Polypeptide for diseases related to angiogenesis and lymphangiogenesis and use thereof |
US11028148B2 (en) * | 2017-09-28 | 2021-06-08 | Geltor, Inc. | Recombinant collagen and elastin molecules and uses thereof |
US11052130B2 (en) | 2015-03-31 | 2021-07-06 | Ildong Pharm Co., Ltd. | Method for treating eye diseases with a fusion protein of a tissue-penetrating peptide and anti-vascular endothelial growth factor |
US11168126B2 (en) | 2019-04-12 | 2021-11-09 | Geltor, Inc. | Recombinant elastin and production thereof |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101551306B1 (en) * | 2015-03-23 | 2015-09-09 | 아주대학교산학협력단 | Neuropilin-1 specific binding peptides and its fusion protein, and use thereof |
WO2017039358A1 (en) * | 2015-09-01 | 2017-03-09 | 일동제약 주식회사 | Pharmaceutical composition containing, as active ingredient, fusion protein in which tumor-penetrating peptide and anti-angiogenesis agent are fused, for preventing and treating cancer or angiogenesis-related diseases |
KR102048474B1 (en) * | 2016-03-29 | 2019-11-26 | 아주대학교산학협력단 | Composition for Inhibiting Tolerance Against Epidermal Growth Factor Receptor Targeting Inhibitor |
WO2017171373A2 (en) * | 2016-03-29 | 2017-10-05 | 아주대학교산학협력단 | Composition for suppressing resistance to egfr-targeting agent |
EP3576782A4 (en) * | 2017-02-02 | 2020-12-30 | Silverback Therapeutics, Inc. | Construct-peptide compositions and methods of use thereof |
KR102382403B1 (en) * | 2017-10-26 | 2022-04-04 | 파인트리 테라퓨틱스 인코포레이티드 | Neuropilin-1 Specific Binding Non-CendR Peptide, Fusion Protein Comprising the Same Fused Thereto and Use Thereof |
CN109082440A (en) * | 2018-08-08 | 2018-12-25 | 华南农业大学 | Rapid Accumulation model and the preparation method and application thereof in a kind of foreign protein body |
WO2020185624A1 (en) * | 2019-03-08 | 2020-09-17 | DrugCendR, Inc. | Low-dose cytokine co-administered with irgd for treating cancer |
KR102459219B1 (en) * | 2022-03-31 | 2022-10-26 | 주식회사 셀아이콘랩 | Novel skin penetrating peptides, composition for skin and mucosal treatment, including the same, and their uses |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001057273A2 (en) * | 2000-02-04 | 2001-08-09 | Aeomica, Inc. | Human genome-derived single exon nucleic acid probes useful for analysis of gene expression in human adult liver |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5639856A (en) * | 1993-09-13 | 1997-06-17 | The Regents Of The University Of California | Semaphorin gene family |
WO2001038491A2 (en) * | 1999-11-08 | 2001-05-31 | The General Hospital Corporation | Novel semaphorin domains |
US6936450B2 (en) * | 2000-04-12 | 2005-08-30 | Compugen Ltd. | Variants of protein kinases |
US7601692B2 (en) * | 2000-11-28 | 2009-10-13 | Compugen Ltd. | MCP-1 splice variants and methods of using same |
US20040142325A1 (en) * | 2001-09-14 | 2004-07-22 | Liat Mintz | Methods and systems for annotating biomolecular sequences |
EP1581790B1 (en) * | 2001-11-08 | 2009-01-07 | The Burnham Institute | Peptides that home to tumor lymphatic vasculature and methods of using same |
US7485414B2 (en) * | 2002-08-30 | 2009-02-03 | Rigel Pharmaceuticals, Inc. | Modulators of angiogenesis |
WO2008002661A2 (en) * | 2006-06-28 | 2008-01-03 | The Board Of Trustees Of The Leland Stanford Junior University | Fusion protein constructs |
ES2620487T3 (en) * | 2007-10-19 | 2017-06-28 | Rappaport Family Institute For Research In The Medical Sciences | Compositions comprising semaforins for use in the treatment of cancer |
SG10201605629VA (en) * | 2008-01-03 | 2016-08-30 | Scripps Research Inst | Antibody targeting through a modular recognition domain |
EP2242764B1 (en) * | 2008-01-18 | 2016-03-30 | Burnham Institute for Medical Research | Methods and compositions related to internalizing rgd peptides |
CN102869384B (en) * | 2009-06-22 | 2016-01-13 | 伯纳姆医学研究所 | Use the method and composition with the peptides and proteins of C-end element |
WO2011066284A1 (en) * | 2009-11-25 | 2011-06-03 | The University Of North Carolina At Chapel Hill | Methods and compositions for the treatment of immune disorders |
AU2014265142A1 (en) * | 2013-05-17 | 2015-12-24 | Medimmune, Llc | Receptors for B7-H4 |
-
2014
- 2014-05-22 KR KR1020140061751A patent/KR101551299B1/en active IP Right Grant
- 2014-05-22 US US14/893,317 patent/US9975933B2/en active Active
- 2014-05-22 EP EP14800779.2A patent/EP3000825A4/en not_active Withdrawn
- 2014-05-22 JP JP2016515272A patent/JP6391676B2/en active Active
- 2014-05-22 WO PCT/KR2014/004571 patent/WO2014189303A1/en active Application Filing
-
2018
- 2018-05-08 JP JP2018090258A patent/JP2018134109A/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001057273A2 (en) * | 2000-02-04 | 2001-08-09 | Aeomica, Inc. | Human genome-derived single exon nucleic acid probes useful for analysis of gene expression in human adult liver |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10364289B2 (en) | 2014-12-03 | 2019-07-30 | Samsung Life Public Welfare Foundation | Antibody binding to neuropilin 1 and use thereof |
US11052130B2 (en) | 2015-03-31 | 2021-07-06 | Ildong Pharm Co., Ltd. | Method for treating eye diseases with a fusion protein of a tissue-penetrating peptide and anti-vascular endothelial growth factor |
US11738064B2 (en) | 2015-03-31 | 2023-08-29 | Ildong Pharm Co., Ltd. | Pharmaceutical composition for preventing and treating eye diseases, containing as active ingredient, fusion protein in which tissue-penetrating peptide and anti-vascular endothelial growth factor preparation are fused |
US11028148B2 (en) * | 2017-09-28 | 2021-06-08 | Geltor, Inc. | Recombinant collagen and elastin molecules and uses thereof |
US11041015B2 (en) | 2017-09-28 | 2021-06-22 | Geltor, Inc. | Recombinant collagen and elastin molecules and uses thereof |
US11180541B2 (en) | 2017-09-28 | 2021-11-23 | Geltor, Inc. | Recombinant collagen and elastin molecules and uses thereof |
US11214609B2 (en) | 2017-09-28 | 2022-01-04 | Geltor, Inc. | Recombinant collagen and elastin molecules and uses thereof |
US11168126B2 (en) | 2019-04-12 | 2021-11-09 | Geltor, Inc. | Recombinant elastin and production thereof |
WO2021068962A1 (en) * | 2019-10-11 | 2021-04-15 | 田中纯美 | Polypeptide for diseases related to angiogenesis and lymphangiogenesis and use thereof |
Also Published As
Publication number | Publication date |
---|---|
KR101551299B1 (en) | 2015-09-10 |
EP3000825A4 (en) | 2017-02-08 |
JP6391676B2 (en) | 2018-09-19 |
KR20140138539A (en) | 2014-12-04 |
JP2016522205A (en) | 2016-07-28 |
US9975933B2 (en) | 2018-05-22 |
JP2018134109A (en) | 2018-08-30 |
EP3000825A1 (en) | 2016-03-30 |
WO2014189303A1 (en) | 2014-11-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9975933B2 (en) | Tumor tissue-penetrating peptide specific to neuropilin and fusion protein having same peptide fused therein | |
US20220041683A1 (en) | Neuropilin-1 Specific Binding Peptide, Fusion Protein Fused with Same, and Use Thereof | |
KR101602870B1 (en) | Method for Cell-penetrating and cytosol-localizing of intact immunoglobulin antibody, and use thereof | |
EP2835380B1 (en) | Method of blocking vascular leakage using an anti-Ang2 antibody | |
KR101602876B1 (en) | Method for inhibiting activated RAS using intact immunoglobulin antibody having Cell-penetrating ability and use thereof | |
KR102163305B1 (en) | Antibody which internalize into the cytosol of cells and binds to inhibit activated Ras and use thereof | |
KR102196450B1 (en) | Anticancer composition containing an anti-Ang2 antibody inducing binding to Tie2 receptor | |
KR20230013143A (en) | Neuropilin-1 Specific Binding Non-CendR Peptide, Fusion Protein Comprising the Same Fused Thereto and Use Thereof | |
KR102054389B1 (en) | Neuropilin-1 Specific Binding Non-CendR Peptide, Fusion Protein Comprising the Same Fused Thereto and Use Thereof | |
CN111065653A (en) | Conditional endocytosis of pegylated reagents by pre-targeted bispecific polyethylene glycol-binding antibodies for diagnostic and therapeutic uses | |
KR20220044918A (en) | Neuropilin-1 Specific Binding Non-CendR Peptide, Fusion Protein Comprising the Same Fused Thereto and Use Thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KIM, YONG SUNG;SHIN, TAE HWAN;KIM, YAE JIN;AND OTHERS;REEL/FRAME:037686/0776 Effective date: 20151224 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
AS | Assignment |
Owner name: PINETREE THERAPEUTICS, INC., MASSACHUSETTS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION;REEL/FRAME:056092/0979 Effective date: 20210428 Owner name: PINETREE THERAPEUTICS, INC., MASSACHUSETTS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION;REEL/FRAME:056093/0102 Effective date: 20210428 |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YR, SMALL ENTITY (ORIGINAL EVENT CODE: M2551); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY Year of fee payment: 4 |